Biocompatible crosslinked polymers with visualization agents

ABSTRACT

Biocompatible crosslinked polymers, and methods for their preparation and use, are disclosed in which the biocompatible crosslinked polymers are formed from water soluble precursors having electrophilic and nucleophilic functional groups capable of reacting and crosslinking in situ. Methods for making the resulting biocompatible crosslinked polymers biodegradable or not are provided, as are methods for controlling the rate of degradation. The crosslinking reactions may be carried out in situ on organs or tissues or outside the body. Applications for such biocompatible crosslinked polymers and their precursors include controlled delivery of drugs, prevention of post-operative adhesions, coating of medical devices such as vascular grafts, wound dressings and surgical sealants. Visualization agents may be included with the crosslinked polymers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/293,892 filed Dec. 2, 2005 issued as U.S. Pat. No. 7,332,566, whichis a continuation of U.S. Pat. No. 7,009,034 filed Nov. 9, 2001, whichis a continuation in part of U.S. patent application Ser. No. 09/147,897filed Aug. 30, 1999, now abandoned, which is a United States nationalstage application of Patent Cooperation Treaty applicationPCT/US97/16897 filed Sep. 22, 1997, which has a priority date based onU.S. applications 60/026,526 filed Sep. 23, 1996; 60/039,904 filed Mar.4, 1997; and 60/040,417 filed Mar. 13, 1997. U.S. patent applicationSer. No. 09/147,897, now abandoned, is also a continuation-in-part ofU.S. patent application Ser. No. 09/454,900 filed Dec. 3, 1999 issued asU.S. Pat. No. 6,566,406, which has a priority date based on U.S. patentapplication 60/110,849 filed Dec. 4, 1998. The present patentapplication claims priority to these other patents and patentapplications which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to biocompatible crosslinkedpolymers, methods for preparing and using same.

BACKGROUND OF THE INVENTION

Almost every surgical treatment carries a risk that bodily tissuesexposed during the course of the surgery will adhere to each other, acondition termed an adhesion. Gynecological and abdominal surgeries, inparticular, are prone to causing adhesions, which often have theappearance of scar-like masses. Adhesions are frequently painful and area significant cause of infertility resulting from gynecologicalsurgeries. Adhesions caused by surgeries are often called surgicaladhesions.

One approach to the treatment of adhesions has been to coat surgicallyexposed tissues with a gel before closing the surgical site. Gels ofvarious types have been used, including suspensions of colloidalparticles, and pastes of natural polymers. Various examples of some ofthese approaches are described in U.S. Pat. Nos. 6,020,326 and5,605,938. Some of these approaches allow for the polymers to be addedto the patient “in situ” in a solution and then chemically reactedinside the patient so that the polymers form covalent crosslinks tocreate a polymer network. This approach lets the polymer be formed in away that closely conforms to the shape of the tissues in the body, asdescribed, for example, in U.S. Pat. Nos. 5,410,016; 5,573,934 and5,626,863.

Hydrogels are especially useful for use in the body because they aremore biocompatible than non-hydrogels and are thus better tolerated inthe body. Besides being useful for post-operative adhesions they can beused for many medical purposes, such as tissue augmentation, medicaldevice coating, surgical sealing, and drug delivery. Examples ofhydrogels formulated for such purposes are found in U.S. Pat. Nos.4,414,976; 4,427,651; 4,925,677; 5,527,856; 5,550,188; and 5,814,621.

Crosslinked polymers have previously been formed using polymers equippedwith either electrophilic or nucleophilic functional groups. Forexample, U.S. Pat. Nos. 5,296,518 and 5,104,909 to Grasel et al.describe the formation of crosslinked polymers from ethylene oxide richprepolymers, wherein a polyisocyanate or low molecular weightdiisocyanate is used as the electrophilic polymer or crosslinker, and apolyoxyethylene based polyol with in situ generated amine groups is usedas the nucleophilic precursor; see also U.S. Pat. Nos. 5,514,379;5,527,856; and 5,550,188.

Polymeric hydrogels, for example, fibrin glue, crosslinked proteins, andcrosslinked polyethylene oxides, are essentially colorless. This problemis often even more acute when the hydrogel is applied as a coating on atissue because tissue coatings conventionally are thin. The resultingcolorless solution or film is therefore difficult to visualize,especially in the typically wet and moist surgical environment. Underlaparoscopic conditions, visibility is even more difficult due to thefact that only a two-dimensional view of the surgical field is availableon the monitor that is used in such procedures.

SUMMARY OF THE INVENTION

The present inventors have realized that use of color in biocompatiblecrosslinked polymers and precursors greatly improves their performancein a surgical environment, especially under minimally invasive surgicalprocedures (MIS), e.g., laparoscopic, endoscopic. Moreover, the bettervisibility available with the use of color also permits efficient use ofmaterials and avoids wastage.

An embodiment of the invention is a hydrogel for use on a substrate suchas a patient's tissue. The hydrogel has water, a biocompatiblevisualization agent, and reactive hydrophilic polymers that form acrosslinked hydrogel after contact with the tissue. The hydrogel coatsthe tissue and forms a coating. The coating may have a free surface. Thevisualization agent is disposed in the hydrogel and reflects or emitslight at a wavelength detectable to a human eye. This feature lets auser applying the hydrogel observe the hydrogel and estimate itsthickness and apply the hydrogel until it reaches a predeterminedthickness.

The hydrophilic polymers may be natural polymers, for example proteinse.g., collagen, fibrinogen, albumin, and fibrin, polysaccharides, orglycosaminoglycans. The polymers can also have a hydrolyticallybiodegradable portion and/or a proteolytically degradable portion. Thepolymers are preferably covalently crosslinked and are crosslinkable viaan electrophilic functional group-nucleophilic functional groupreaction. An embodiment of the invention is a hydrogel that is coatedonto a tissue and has a maximum thickness of between 0.1 to 10.0 mm.

Preferred biocompatible visualization agents are FD&C Blue #1, #2, #3,D&C Green #6, and methylene blue. The visualization agent may also be afluorescent molecule. The visualization agent is preferably notcovalently linked to the hydrogel.

Methods for using the polymeric compositions to coat a tissue includemixing hydrophilic precursor polymers with chemically distinct reactivefunctional groups such that they form crosslinks vianucleophilic-electrophilic reaction after mixing and contact with thetissue. The polymers crosslink to form a biodegradable hydrogel. Apreferred application is to prevent surgical adhesions by applying thehydrogel as a coating on a tissue substrate and maintaining anothersurface of the hydrogel as a free surface. A visualization agent ispreferably included so that the visualization agent is disposed withinthe hydrogel and reflects or emits light at a wavelength detectable to ahuman eye. A preferred method of use is to form a hydrogel on the tissueuntil the color and/or color intensity of the hydrogel indicates that apredetermined thickness of hydrogel has been deposited on the tissue.

An embodiment of the invention is a polymeric product made by a processof mixing hydrophilic polymers having nucleophilic functional groupswith hydrophilic polymers having electrophilic functional groups suchthat they form a mix that crosslinks after contact with the tissue of apatient to form a biodegradable hydrogel that coats a tissue. In manyapplications it is desirable to also have a free surface. The hydrogelpreferably contains a visualization agent in the mix of reactiveprecursor species so that the visualization agent is disposed within theinterior and reflects or emits light at a wavelength detectable to ahuman eye.

An embodiment of the invention is a kit having a biocompatiblevisualization agent, at least two chemically distinct reactive precursorspecies, and instructions for using the visualization agent and thereactive precursor species such that the reactive precursor species maybe combined to form crosslinked hydrophilic polymers that form abiodegradable hydrogel. In another embodiment, the visualization agentis premixed with one of the reactive precursor species.

It is an object of the present invention to provide biocompatiblecrosslinked polymers and methods for their preparation and use, in whichthe biocompatible crosslinked polymers are formed without using freeradical chemistry, and are formed using at least one non-toxic smallmolecule precursor.

It is another object of this invention to provide such biocompatiblecrosslinked polymers and methods for their preparation and use, in whichthe biocompatible crosslinked polymers are formed from aqueoussolutions, preferably under physiological conditions.

It is still another object of this invention to provide suchbiocompatible crosslinked polymers and methods for their preparation anduse, in which the biocompatible crosslinked polymers are formed in vivo.

It is a still further object of this invention to provide suchbiocompatible crosslinked polymers and methods for their preparation anduse, in which the biocompatible crosslinked polymers are biodegradable.

Another object of this invention is to provide such biocompatiblecrosslinked polymers and methods for their preparation and use, in whichthe biocompatible crosslinked polymers, their precursors, or both arecolored.

Another object of this invention is to provide methods for preparingtissue conforming, biocompatible crosslinked polymers in a desirableform, size and shape.

Another object of this invention is to provide methods for usingbiocompatible crosslinked polymers to form medically useful devices orimplants for use as surgical adhesion prevention barriers, asimplantable wound dressings, as scaffolds for cellular growth for tissueengineering or as surgical tissue adhesives or sealants.

Another object of this invention is to provide methods for usingbiocompatible crosslinked polymers to form medically useful devices orimplants that can release bioactive compounds in a controlled manner forlocal, systemic, or targeted drug delivery.

Another object of this invention is to provide methods and compositionsfor producing composite biomaterials comprising fibers or particulatesmade of biodegradable biocompatible crosslinked polymers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-E depict electrophilic functional group water soluble andbiodegradable crosslinkers or functional polymers, which can becrosslinked with appropriate nucleophilic functional group precursors.

FIG. 2F-J depict nucleophilic water soluble and biodegradablecrosslinkers or functional polymers, which can be crosslinked withappropriate electrophilic precursors.

FIG. 3K-O depict electrophilic water soluble and biodegradablecrosslinkers or functional polymers, which can be crosslinked withappropriate nucleophilic functional group precursors, wherein either thebiodegradable linkages or the functional groups are selected so as tomake the precursor water soluble.

FIG. 4P-T depict nucleophilic functional group water solublecrosslinkers or functional polymers, which can be crosslinked withappropriate electrophilic functional group precursors, and which are notbiodegradable.

FIG. 5U-Y depict electrophilic water soluble crosslinkers or functionalpolymers, which can be crosslinked with appropriate nucleophilicfunctional group precursors, and which are not biodegradable.

FIG. 6 depicts the preparation of an electrophilic water solublecrosslinker or functional polymer using carbodiimide (“CDI”) activationchemistry, its crosslinking reaction with a nucleophilic water solublefunctional polymer to form a biocompatible crosslinked polymer product,and the hydrolysis of that biocompatible crosslinked polymer to yieldwater soluble fragments.

FIG. 7 depicts the use of sulfonyl chloride activation chemistry toprepare an electrophilic functional polymer.

FIG. 8 depicts the preparation of an electrophilic water solublecrosslinker or functional polymer using N-hydroxysuccinimide (“NHS”)activation chemistry, its crosslinking reaction with a nucleophilicwater soluble functional polymer to form a biocompatible crosslinkedpolymer product, and the hydrolysis of that biocompatible crosslinkedpolymer to yield water soluble fragments.

FIG. 9 depicts preferred NHS esters for use in the invention.

FIG. 10 shows the N-hydroxysulfosuccinimide (“SNHS”) activation of atetrafunctional sugar-based water soluble synthetic crosslinker and itscrosslinking reaction with 4-arm amine terminated polyethylene glycol toform a biocompatible crosslinked polymer product, and the hydrolysis ofthat biocompatible crosslinked polymer to yield water soluble fragments.

FIG. 11 shows the variation in gelation time with the number of aminogroups for the reaction of 4 arm 10 kDa succinimidyl glutarate PEG(“SG-PEG”) with di-,tri- or tetra-lysine.

FIG. 12 shows the variation in gelation time with the solution age ofthe electrophilic functional polymer.

FIG. 13 shows the variation in gelation time with the concentration ofbiocompatible crosslinked polymer precursors, and with the solution ageof the 4 arm 10 kDa carboxymethyl-hydroxybutyrate-N-hydroxysuccinimidylPEG (“CM-HBA-NS”) electrophilic functional polymer.

FIG. 14 shows the variation in degradation time with the concentrationof biocompatible crosslinked polymer.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have realized that use of color in biocompatiblecrosslinked polymers and/or reactive precursor species improves theperformance of crosslinked networks of polymers and/or reactiveprecursor species in a surgical environment, especially for minimallyinvasive surgical (MIS) procedures. Many applications have the bestresults when an appropriate or predetermined amount of hydrogel isdelivered to the surgical environment, for example when applied to thesurface of a substrate such as a tissue. A hydrogel that is too thickmay reduce efficiency or interfere with other surgical aspects. Forexample, if a hydrogel is applied too thickly, it could interfere withclosure of the wound or interfere with tissue movement, e.g., inintestinal applications. A hydrogel that is too thin will not serve itspurpose, e.g., providing a barrier that prevents surgical adhesions orprovides a strong seal against fluid leakage. The introduction of avisualization agent allows the user to determine the thickness of theapplied hydrogel. The visualization agent is preferably an agent thatprovides a color that is visible to the human eye, e.g., a color that isdetected visually by the user or detected by a video camera and relayedto a video screen observed by the user.

Conventional polymeric hydrogels may sometimes have a faint inherentcolor or develop a faint color as a result of chemical activity, buttheir lack of color makes a layer of a hydrogel very difficult to seeafter it has been applied to a tissue. Hydrogels have sometimes beenmixed with image contrast agents to increase their visibility formedical imaging devices such as X-ray or magnetic resonance imaging(MRI) machines, as in, for example, U.S. Pat. No. 5,514,379. Colorantshave also been used for hydrogels injected into bodily tissues, forexample in U.S. Pat. Nos. 5,514,379 and 6,124,273.

The use of a visualization agent is especially preferred when a hydrogelis used to coat a substrate. A substrate coating surface is a surface ofa hydrogel that contacts a substrate and, in the region of contact, isessentially in continuous contact with that substrate. Although somesmall portions of the coating or substrate may not be in contact, thecontact is intimate. A substrate coating surface can be formed when thehydrogel crosslinks after contacting the substrate surface because thecontact before crosslinking allows the hydrogel precursors to mix andconform to the shape of the substrate. A preformed hydrogel materialgenerally does not have a substrate coating surface. A preferredsubstrate is a tissue of a patient.

A hydrogel with a substrate coating surface preferably also has a freesurface when the hydrogel is used for prevention of adhesions. Thehydrogel is applied to a tissue and crosslinks while having one freesurface that is not adherent to any tissue but is instead freely movablerelative to any tissues that it may subsequently contact. The freesurface prevents the coated tissue from contact with other tissues anddoes not prevent the movement of other tissues so that protection andfree movement are optimal. In this situation, a user that applies thehydrogel may observe the hydrogel by looking through the free surfaceinto the hydrogel and at the coated tissue. A visualization agent in thehydrogel makes the hydrogel change in its appearance until the userdetermines that the thickness of the hydrogel is sufficient. Forexample, a blue dye in the hydrogel makes the hydrogel increasinglyopaque as the thickness of the hydrogel increases.

It is preferable to provide color by adding a colored visualizationagent to the hydrogel precursors before crosslinking. The coloring agentis thus present in a premixed amount that is already selected for theapplication. A preferred embodiment of the invention uses biocompatiblecrosslinked polymers formed from the reaction of precursors havingelectrophilic functional group and nucleophilic functional groups. Theprecursors are preferably water soluble, non-toxic and biologicallyacceptable.

Preferably, at least one of the precursors is a small molecule of about1000 Da or less, and is referred to as a “crosslinker”. The crosslinkerpreferably has a solubility of at least 1 g/100 mL in an aqueoussolution. A crosslinked molecule may be crosslinked via an ionic orcovalent bond, a physical force, or other attraction. Preferably, atleast one of the other precursors is a macromolecule, and is referred toas a “functional polymer”. The macromolecule, when reacted incombination with a crosslinker, is preferably at least five to fiftytimes greater in molecular weight than the small molecule crosslinkerand is preferably less than about 60,000 Da. A more preferred range is amacromolecule that is seven to thirty times greater in molecular weightthan the crosslinker and a most preferred range is about ten to twentytimes difference in weight. Further, a macromolecular molecular weightof 5,000 to 50,000 is preferred, a molecular weight of 7,000 to 40,000is more preferred and a molecular weight of 10,000 to 20,000 is mostpreferred. The term polymer, as used herein, means a molecule formed ofat least three repeating groups. The term “reactive precursor species”means a polymer, functional polymer, macromolecule, small molecule, orcrosslinker that can take part in a reaction to form a network ofcrosslinked molecules, e.g., a hydrogel.

An embodiment of the invention is a hydrogel for use on a patient'stissue that has water, a biocompatible visualization agent, andcrosslinked hydrophilic polymers that form a hydrogel after contact withthe tissue. The hydrogel coats the tissue and also has a free surface.The visualization agent reflects or emits light at a wavelengthdetectable to a human eye so that a user applying the hydrogel canobserve the gel and also estimate its thickness.

Natural polymers, for example proteins or glycosaminoglycans, e.g.,collagen, fibrinogen, albumin, and fibrin, may be crosslinked usingreactive precursor species with electrophilic functional groups. Naturalpolymers are proteolytically degraded by proteases present in the body.Synthetic polymers and reactive precursor species are preferred,however, and may have electrophilic functional groups that arecarbodiimidazole, sulfonyl chloride, chlorocarbonates,n-hydroxysuccinimidyl ester, succinimidyl ester or sulfasuccinimidylesters. The term synthetic means a molecule that is not found in nature,e.g., polyethylene glycol. The nucleophilic functional groups may be,for example, amine, hydroxyl, carboxyl, and thiol. The polymerspreferably have a polyalkylene glycol portion. More preferably they arepolyethylene glycol based. The polymers preferably also have ahydrolytically biodegradable portion or linkage, for example an ester,carbonate, or an amide linkage. Several such linkages are well known inthe art and originate from alpha-hydroxy acids, their cyclic dimmers, orother chemical species used to synthesize biodegradable articles, suchas, glycolide, dl-lactide, l-lactide, caprolactone, dioxanone,trimethylene carbonate or a copolymer thereof. A preferred embodimenthas reactive precursor species with two to ten nucleophilic functionalgroups each and reactive precursor species with two to ten electrophilicfunctional groups each. The hydrophilic species are preferably syntheticmolecules.

Preferred biocompatible visualization agents are FD&C BLUE #1, FD&C BLUE#2, and methylene blue. These agents are preferably present in the finalelectrophilic-nucleophilic reactive precursor species mix at aconcentration of more than 0.05 mg/ml and preferably in a concentrationrange of at least 0.1 to about 12 mg/ml, and more preferably in therange of 0.1 to 4.0 mg/ml, although greater concentrations maypotentially be used, up to the limit of solubility of the visualizationagent. These concentration ranges were found to give a color to thehydrogel that was desirable without interfering with crosslinking times(as measured by the time for the reactive precursor species to gel). Thevisualization agent may also be a fluorescent molecule. Thevisualization agent is preferably not covalently linked to the hydrogel.

An embodiment of the invention is a hydrogel that is coated onto atissue and generally has at least a portion with a thickness of between0.8 to 12.0 mm. One technique for measuring the thickness is to create ahydrogel on a test surface and use a micrometer to measure thicknessesat various points. Alternatively, a calibrated videomicroscopic imagecould be used. The preferred thickness depends on the medicalapplication but a preferred thickness for prevention of surgicaladhesions is about 0.5 to 10.0 mm, and more preferably about 0.8 to 5 mmand even more preferably about 1-3 mm.

A preferred method of use is to form a hydrogel on the tissue until thecolor of the hydrogel indicates that a predetermined thickness ofhydrogel has been deposited on the tissue. The deposition of theprecursors that result in formation of the hydrogel may be by spraying,dripping, or delivery via a catheter. The user may apply the hydrogel toa test surface with a color that resembles the surface that the usercontemplates using and observe the color that results when the hydrogelreaches a desired thickness that the user has predetermined. In use, theuser applies the hydrogel until the desired color is reached. A typicalpatient's tissue has a pinkish appearance and the microvasculature canbe observed as thin lines. One embodiment is to introduce aconcentration of visualization agent into the hydrogel so that the userapplies the hydrogel until the microvasculature is no longer visiblethrough the hydrogel, at which point the hydrogel is a desiredthickness. Another suitable method is to apply the hydrogel until theunderlying tissue is obscured. An appropriately selected concentrationof visualization agent is used so that the hydrogel obscures the tissuefeatures when the hydrogel achieves a predetermined thickness. Thepredetermined thickness is chosen to correspond to the particularapplication. In these thickness evaluation approaches, a concentrationthat is too low will result in a hydrogel that is too thick and aconcentration that is too high will result in a hydrogel that is toothin. Thus, the visualization agent allows the user to ascertain thepresence of the hydrogel on the surface and also gain feedback on theappropriate thickness, preferably in combination with instructionsprovided as part of a kit. In some embodiments, suitable approaches canbe used with visualization agents and polymers that crosslink by, forexample, free radical polymerization, electrophilic functionalgroup-nucleophilic functional group interaction.

An embodiment of the invention is a method of a user applying a hydrogelcoating to a substrate and selecting a visually observable visualizationagent to observe the hydrogel coating. The user may use visualizationagents to see the hydrogel with the human eye or with the aid of animaging device that detects visually observable visualization agents,e.g., a videocamera. A visually observable visualization agent is anagent that has a color detectable by a human eye. A characteristic ofproviding imaging to an X-ray or MRI machine is not a characteristicsufficient to establish function as a visually observable visualizationagent. An alternative embodiment is a visualization agent that may notnormally be seen by the human eye but is detectable at a differentwavelength, e.g., the infra red or ultraviolet, when used in combinationwith a suitable imaging device, e.g., a videocamera.

A coating has a surface that can be viewed for use with a visuallyobservable visualization agent. In contrast, a hydrogel injected into ablood vessel, muscle, or other tissue has essentially no surface forviewing a visualization agent because its surface area is essentiallyengaged with tissues of the patient. Further, polymers injected into atissue lack a surface that is disposed on the surface of a tissue and donot provide a means for a user to control the thickness of the coatingon the surface of the tissue. Hydrogels that are merely injected into apatient's body would not be equivalent to embodiments of the presentinvention that involve a hydrogel coating on a substrate and areinoperative for embodiments of the invention that entail use of avisualization agent in a hydrogel coating.

An embodiment of the invention involves a mixture or a process of mixinghydrophilic reactive precursor species having nucleophilic functionalgroups with hydrophilic reactive precursor species having electrophilicfunctional groups such that they form a mixture that crosslinks quicklyafter contact with the tissue of a patient to form a biodegradablehydrogel that coats and adheres to a tissue. This may be achieved bymaking reactive precursor species that crosslink quickly after mixing.Hydrophilic reactive precursor species can be dissolved in bufferedwater such that they provide low viscosity solutions that readily mixand flow when contacting the tissue. As they flow across the tissue,they conform to the shape of the small features of the tissue such asbumps, crevices and any deviation from molecular smoothness. If thereactive precursor species are too slow to crosslink, they will flow offthe tissue and away into other portions of the body with the result thatthe user will be unable to localize the hydrogel on the desired tissue.Without limiting the invention to a particular theory of operation, itis believed that reactive precursor species that crosslink appropriatelyquickly after contacting a tissue surface will form a three dimensionalstructure that is mechanically interlocked with the coated tissue. Thisinterlocking contributes to adherence, intimate contact, and essentiallycontinuous coverage of the coated region of the tissue.

Adherence is important for medical applications that require a coating,e.g., for prevention of adhesions, since a user must be able to placethe hydrogel in the portions of the patient that are needful, forexample, around an ovary or surrounding an intestine. Further, thehydrogel must remain on the intended tissue or it will be unable toprovide a prophylactic barrier. The hydrogels of the invention have goodadhesion onto tissue and are useful for all applications whereinsurgical glues have previously been used. For example, sealing of thedura mater of the brain to prevent leakage of cerebrospinal fluid may beaccomplished with combinations of reactive precursor species describedherein by using reactive precursor species with nucleophilic functionalgroups for mixing with hydrophilic reactive precursor species havingelectrophilic functional groups to form a mix that crosslinks quicklyafter contact with the tissue of a patient, e.g., the dura mater, toform a hydrogel that coats a tissue.

A simple dip test shows that a hydrogel has adherence. To perform thistest, a gel of about 5×5 centimeters in length×width and about 4 to 10mm in thickness is formed on a substrate, the hydrogel is immersed inwater or physiological saline for five minutes, removed, and tilted toan angle of 90 degrees above horizontal, and dipped into and out of avessel of physiological saline five times at a rate of about 10 mm persecond so that the hydrogel passes through the air-water interface tentimes. Then the substrate is rotated about 90 degrees so that thesubstrate is approximately horizontal and the hydrogel is below thesubstrate. The substrate is left in this position for five minutes. Thegel passes the dip test if less than about 1 square centimeter of thegel is then observed to be separated from the substrate. If thesubstrate lacks stiffness, it may be affixed to a stiff support so thatit may be tested. Physiological saline, in the context of the dip test,means a saline solution with an approximately physiological osmolarityand a pH of 7.0-7.4 at room temperature that is customarily used in cellculture, for example, phosphate buffered saline. As used herein, the gelhas adherence to a substrate if it passes the dip test.

Suitable crosslinking times vary for different applications. In mostapplications, the crosslinking reaction leading to gelation occurswithin about 10 minutes, more preferably within about 2 minutes, evenmore preferably within 10 seconds. In the case of most surgical adhesionprevention applications, it is preferable to use a hydrogel thatcrosslinks in less than about 10 seconds and more preferably in about2-4 seconds in order to allow a user to make multiple passes with ahydrogel applicator tool such as a sprayer; see, for example commonlyassigned U.S. Pat. Nos. 6,179,862; 6,165,201; 6,152,943; and U.S. patentapplication Ser. Nos. 09/687,588, which are hereby incorporated hereinby reference. In the case of tissues that can be accessed onlyindirectly, longer times are most preferable to allow the gel a longertime to flow into the inaccessible space. For example, application of anadhesion barrier in and around the spinal cord and exiting nerve rootsfollowing spine surgery may require several extra seconds to penetratearound the complex geometry of the tissues so that a preferred time isbetween about 5 and about 90 seconds and more preferably between about10 and about 30 seconds. The Examples herein describe a variety ofreactive precursor species and methods of making reactive precursorspecies that may be mixed to provide crosslinked networks that crosslinkquickly after mixing such that one skilled in these arts will understandhow to make the materials of the invention after reading thisdisclosure.

Functional Groups

Each precursor is multifunctional, meaning that it comprises two or moreelectrophilic or nucleophilic functional groups, such that anucleophilic functional group on one precursor may react with anelectrophilic functional group on another precursor to form a covalentbond. At least one of the precursors comprises more than two functionalgroups, so that, as a result of electrophilic-nucleophilic reactions,the precursors combine to form crosslinked polymeric products. Suchreactions are referred to as “crosslinking reactions”.

Preferably, each precursor comprises only nucleophilic or onlyelectrophilic functional groups, so long as both nucleophilic andelectrophilic precursors are used in the crosslinking reaction. Thus,for example, if a crosslinker has nucleophilic functional groups such asamines, the functional polymer may have electrophilic functional groupssuch as N-hydroxysuccinimides. On the other hand, if a crosslinker haselectrophilic functional groups such as sulfosuccinimides, then thefunctional polymer may have nucleophilic functional groups such asamines or thiols. Thus, functional polymers such as proteins, poly(allylamine), or amine-terminated di- or multifunctional poly(ethylene glycol)(“PEG”) can be used.

Water Soluble Cores

The precursors preferably have biologically inert and water solublecores. When the core is a polymeric region that is water soluble,preferred polymers that may be used include: polyether, for example,polyalkylene oxides such as polyethylene glycol (“PEG”), polyethyleneoxide (“PEO”), polyethylene oxide-co-polypropylene oxide (“PPO”),co-polyethylene oxide block or random copolymers, and polyvinyl alcohol(“PVA”); poly(vinyl pyrrolidinone) (“PVP”); poly(amino acids); dextranand proteins such as albumin. The polyethers and more particularlypoly(oxyalkylenes) or poly(ethylene glycol) or polyethylene glycol areespecially preferred. When the core is small molecular in nature, any ofa variety of hydrophilic functionalities can be used to make theprecursor water soluble. For example, functional groups like hydroxyl,amine, sulfonate and carboxylate, which are water soluble, maybe used tomake the precursor water soluble. In addition, N-hydroxysuccinimide(“NHS”) ester of subaric acid is insoluble in water, but by adding asulfonate group to the succinimide ring, the NHS ester of subaric acidmay be made water soluble, without affecting its reactivity towardsamine groups.

Biodegradable Linkages

If it is desired that the biocompatible crosslinked polymer bebiodegradable or absorbable, one or more precursors having biodegradablelinkages present in between the functional groups may be used. Thebiodegradable linkage optionally also may serve as the water solublecore of one or more of the precursors. In the alternative, or inaddition, the functional groups of the precursors may be chosen suchthat the product of the reaction between them results in a biodegradablelinkage. For each approach, biodegradable linkages may be chosen suchthat the resulting biodegradable biocompatible crosslinked polymer willdegrade or be absorbed in a desired period of time. Preferably,biodegradable linkages are selected that degrade under physiologicalconditions into non-toxic products.

The biodegradable linkage may be chemically or enzymaticallyhydrolyzable or absorbable. Illustrative chemically hydrolyzablebiodegradable linkages include polymers, copolymers and oligomers ofglycolide, dl-lactide, l-lactide, caprolactone, dioxanone, andtrimethylene carbonate. Illustrative enzymatically hydrolyzablebiodegradable linkages include peptidic linkages cleavable bymetalloproteinases and collagenases. Additional illustrativebiodegradable linkages include polymers and copolymers of poly(hydroxyacid)s, poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.

The biodegradable linkage may be chemically or enzymaticallyhydrolyzable or absorbable. Illustrative chemically hydrolyzablebiodegradable linkages include polymers, copolymers and oligomers ofglycolide, dl-lactide, l-lactide, caprolactone, dioxanone, andtrimethylene carbonate. Illustrative enzymatically5 hydrolyzablebiodegradable linkages include peptidic linkages cleavable bymetalloproteinases and collagenases. Additional illustrativebiodegradable linkages include polymers and copolymers of poly(hydroxyacid)s, poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.

Visualization Agents

Where convenient, the biocompatible crosslinked polymer or precursorsolutions (or both) may contain visualization agents to improve theirvisibility during surgical procedures. Visualization agents areespecially useful when used in MIS procedures, due among other reasonsto their improved visibility on a color monitor.

Visualization agents may be selected from among any of the variousnon-toxic colored substances suitable for use in medical implantablemedical devices, such as FD&C BLUE dyes 3 and 6, eosin, methylene blue,indocyanine green, or colored dyes normally found in synthetic surgicalsutures. The preferred color is green or blue because it has bettervisibility in presence of blood or on a pink or white tissue background.Red is the least preferred color, when used on a highly vascularizedtissue that is red in color. However, red may be suitable when theunderlying tissue is white, for example the cornea.

The visualization agent may be present with either reactive precursorspecies, e.g., a crosslinker or functional polymer solution. Thepreferred colored substance may or may not become chemically bound tothe hydrogel.

The visualization agent may be used in small quantities, preferably lessthan 1% weight/volume, more preferably less that 0.01% weight/volume andmost preferably less than 0.001% weight/volume concentration.

Additional visualization agents may be used, such as fluorescent (e.g.,green or yellow fluorescent under visible light) compounds (e.g.,fluorescein or eosin), x-ray contrast agents (e.g., iodinated compounds)for visibility under x-ray imaging equipment, ultrasonic contrastagents, or MRI contrast agents (e.g., Gadolinium containing compounds).

Visually observable visualization agents are preferred. Wavelengths oflight from about 400 to 750 nm are observable to the human as colors (R.K. Hobbie, Intermediate Physics for Medicine and Biology, 2^(nd) Ed.,pages 371-373). Blue color is perceived when the eye receives light thatis predominantly from about 450 to 500 nm in wavelength and green isperceived at about 500 to 570 nm (Id.). The color of an object istherefore determined by the predominant wavelength of light that itreflects or emits. Further, since the eye detects red or green or blue,a combination of these colors may be used to simulate any other colormerely by causing the eye to receive the proportion of red, green, andblue that is perceived as the desired color by the human eye. Blue andgreen visualization agents are preferred since they are most readilyvisible when observing in situ crosslinking due to the approximately redcolor of the background color of tissue and blood. The color blue, asused herein, means the color that is perceived by a normal human eyestimulated by a wavelength of about 450 to 500 nm and the color green,as used herein, means the color that is perceived by a normal human eyestimulated by a wavelength of about 500 to 570 nm.

Crosslinking Reactions

The crosslinking reactions preferably occur in aqueous solution underphysiological conditions. More preferably the crosslinking reactionsoccur “in situ”, meaning they occur at local sites such as on organs ortissues in a living animal or human body. More preferably thecrosslinking reactions do not release heat of polymerization. Preferablythe crosslinking reaction leading to gelation occurs within about 10minutes, more preferably within about 2 minutes, more preferably withinabout one minute, and most preferably within about 30 seconds. When itis desirable to build up a coating on a convex surface, the crosslinkingreaction preferably occurs within about 2 minutes, more preferably in30-60 seconds, and most preferably in 2-4 seconds.

Certain functional groups, such as alcohols or carboxylic acids, do notnormally react with other functional groups, such as amines, underphysiological conditions (e.g., pH 7.2-11.0, 37° C.). However, suchfunctional groups can be made more reactive by using an activating groupsuch as N-hydroxysuccinimide. Several methods for activating suchfunctional groups are known in the art. Preferred activating groupsinclude carbonyldiimidazole, sulfonyl chloride, aryl halides,sulfosuccinimidyl esters, N-hydroxysuccinimidyl ester, succinimidylester, epoxide, aldehyde, maleimides, imidoesters and the like. TheN-hydroxysuccinimide esters or N-hydroxysulfosuccinimide groups are themost preferred groups for crosslinking of proteins or aminefunctionalized polymers such as amino terminated polyethylene glycol(“APEG”).

FIGS. 1 to 5 illustrate various embodiments of preferred crosslinkersand functional polymers.

FIG. 1 illustrates possible configurations of degradable electrophiliccrosslinkers or functional polymers. The biodegradable regions arerepresented by

 the functional groups are represented by

 and the inert water soluble cores are represented by (——). Forcrosslinkers, the central core is a water soluble small molecule and forfunctional polymers the central core is a water soluble polymer ofnatural or synthetic origin.

When Structure A in FIG. 1 is a functional polymer, it is a linear watersoluble and biodegradable functional polymer, end-capped with twofunctional groups (e.g., N-hydroxysuccinimide ester or NHS, epoxide orsimilar reactive groups). The water soluble core may be a polyalkyleneoxide, preferably polyethylene glycol block copolymer, and it isextended with at least one biodegradable linkage between it and eachterminal functional group. The biodegradable linkage may be a singlelinkage or copolymers or homopolymers of absorbable polymers such aspolyhydroxy acids or polylactones.

When Structure B in FIG. 1 is a functional polymer it is a branched orstar shaped biodegradable functional polymer which has an inert polymerat the center. Its inert and water soluble core is terminated witholigomeric biodegradable extensions, which in turn are terminated withreactive functional groups.

When Structures C and D in FIG. 1 are functional polymers, they aremultifunctional 4 arm biodegradable functional polymers. This polymeragain has a water-soluble soluble core at the center, which is a 4 arm,tetrafunctional polyethylene glycol (Structure C) or block copolymer ofPEO-PPO-PEO such as TETRONIC 908 (Structure D) which is extended with bysmall oligomeric extensions of biodegradable polymer to maintain watersolubility and terminated with reactive functional end-groups such asCDI or NHS.

When Structure E in FIG. 1 is a functional polymer, it is amultifunctional star or graft type biodegradable polymer. This polymerhas a water-soluble polymer like polyethylene oxide, polyvinyl alcoholor poly(vinyl pyrrolidinone) at the core which is completely orpartially extended with biodegradable polymer. The biodegradable polymeris terminated with reactive end groups.

Structures A-E in FIG. 1 need not have polymeric cores and may be smallmolecule crosslinkers. In that case, the core may comprise a smallmolecule like ethoxylated glycerol, inositol, trimethylolpropane etc. toform the resultant crosslinker. In addition, Structures A-E in FIG. 1need not have polymeric biodegradable extensions, and the biodegradableextensions may consist of small molecules like succinate or glutarate orcombinations of 2 or more esters, such as glycolate/2-hydroxybutyrate orglycolate/4-hydroxyproline, etc. A dimer or trimer of 4-hydroxyprolinemay be used not only to add degradability, but also to add nucleophilicfunctional group reactive sites via the pendant primary amines which arepart of the hydroxyproline moiety.

Other variations of the core, the biodegradable linkage, and theterminal electrophilic group in Structures A-E in FIG. 1 may beconstructed, so long as the resulting functional polymer has theproperties of low tissue toxicity, water solubility, and reactivity withnucleophilic functional groups.

FIG. 2 illustrates various embodiments of nucleophilic biodegradablewater soluble crosslinkers and functional polymers suitable for use withelectrophilic functional polymers and crosslinkers described herein.

The biodegradable regions are represented by

 the functional groups are represented by

 and the inert water soluble cores are represented by (——). Forcrosslinkers, the central core is a water soluble small molecule and forfunctional polymers the central core is a water soluble polymer ofnatural or synthetic origin.

When Structure F in FIG. 2 is a functional polymer, it is a linear watersoluble biodegradable polymer terminated with reactive functional groupslike primary amine. The linear water-soluble core is a polyalkyleneoxide, preferably polyethylene glycol block copolymer, which is extendedwith the biodegradable region which is a copolymer or homopolymers ofpolyhydroxy acids or polylactones. This biodegradable polymer isterminated with primary amines.

When Structure G in FIG. 2 is a functional polymer, it is a branched orstar shaped biodegradable polymer which has an inert polymer at thecenter. The inert polymer is extended with single or oligomericbiodegradable extensions which are terminated with reactive functionalgroups.

When Structures H and I in FIG. 2 are functional polymers, they aremultifunctional 4 arm biodegradable polymers. These polymers again havewater-soluble cores at their center which are either a 4 arm,tetrafunctional polyethylene glycol (Structure H) or a block copolymerof PEO-PPO-PEO such as TETRONIC 908 (Structure I), extended with smalloligomeric extensions of biodegradable polymers to maintain watersolubility, and terminated with functional groups such as amines andthiols.

When Structure J in FIG. 2 is a functional polymer, it is amultifunctional star or graft type biodegradable polymer. This polymerhas a water soluble polymer like polyethylene oxide, polyvinyl alcoholor poly(vinyl pyrrolidinone) at the core which is completely orpartially extended with biodegradable polymer. The biodegradable polymeris terminated with reactive end groups.

Structures F-J in FIG. 2 need not have polymeric cores and may be smallmolecule crosslinkers. In that case, the core may comprise a smallmolecule like ethoxylated glycerol, inositol, trimethylolpropane etc. toform the resultant crosslinker.

Other variations of the core, the biodegradable linkage, and theterminal nucleophilic functional group in Structures F-J in FIG. 2 maybe constructed, so long as the resulting functional polymer has theproperties of low tissue toxicity, water solubility, and reactivity withelectrophilic functional groups.

FIG. 3 illustrates configurations of water-soluble electrophiliccrosslinkers or functional polymers where the core is biodegradable. Thebiodegradable regions are represented by

 and the functional groups are represented by

. The biodegradable core is terminated with a reactive functional groupthat is also water solubilizing, such a N-hydroxysulfosuccinimide ester(“SNHS”) or N-hydroxyethoxylated succinimide ester (“ENHS”).

Structure K in FIG. 3 depicts a difunctional biodegradable polymer oroligomer terminated with SNHS or ENHS. The oligomers and polymers may bemade of a poly(hydroxy acid) such as poly(lactic acid), which isinsoluble in water. However, the terminal carboxylic acid group of theseoligomers or polymers can be activated with N-hydroxysulfosuccinimideester (“SNHS”) or N-hydroxyethoxylated succinimide ester (“ENHS”)groups. An ionic group, like a metal salt (preferably sodium salt) ofsulfonic acid, or a nonionic group, like a polyethylene oxide on thesuccinimide ring, provides water-solubility while the NHS ester provideschemical reactivity towards amines. The sulfonate groups (sodium salts)or ethoxylated groups on the succinimide ring solubilize the oligomer orpolymer without appreciably inhibiting reactivity towards amine groups.

Structures L-O in FIG. 3 represent multi-branched or graft typestructures with terminal SNHS or ENHS group. The cores may comprisevarious non-toxic polyhydroxy compounds like sugars (xylitol,erythritol), glycerol, trimethylolpropane, which have been reacted withanhydrides such as succinic or glutaric anhydrides. The resultant acidgroups were then activated with SNHS or ENHS groups to form watersoluble crosslinkers or functional polymers.

FIG. 4 illustrates various nucleophilic functional polymers orcrosslinkers that are not biodegradable. The nucleophilic functionalgroups are represented by

 and the inert water-soluble cores are represented by (——). Forcrosslinkers, the central core is a water-soluble small molecule and forfunctional polymers the central core is a water soluble polymer ofnatural or synthetic origin.

When Structure P in FIG. 4 is a functional polymer it may be awater-soluble linear polymer such as polyethylene glycol terminated withreactive end group such as primary amines and thiols. Such polymers arecommercially available from Sigma (Milwaukee, Wis.) and ShearwaterPolymers (Huntsville, Ala.). Some other preferred difunctional polymersare PPO-PEO-PPO block copolymers such as PLURONIC F68 terminated withamine groups. PLURONIC or TETRONIC polymers are normally available withterminal hydroxyl groups. The hydroxyl groups are converted into aminegroups by methods known in the art.

When Structures Q-T in FIG. 4 are functional polymers they may bemultifunctional graft or branch type water soluble copolymers withterminal amine groups.

Structures P-T in FIG. 4 need not have polymeric cores and may be smallmolecule crosslinkers. In that case, the core may comprise a smallmolecule like ethoxylated glycerol, inositol, trimethylolpropane,dilysine etc. to form the resultant crosslinker.

Other variations of the core and the terminal nucleophilic functionalgroup in Structure P-T in FIG. 4 may be employed, so long as theproperties of low tissue toxicity, water solubility, and reactivity withelectrophilic functional groups are maintained.

FIG. 5 illustrates various electrophilic functional polymers orcrosslinkers that are not biodegradable. The electrophilic functionalgroups are represented by

 and the inert water soluble cores are represented by (——). Forcrosslinkers, the central core is a water soluble small molecule and forfunctional polymers the central core is a water soluble polymer ofnatural or synthetic origin.

When Structure U is a functional polymer, it may be a water-solublepolymer such as polyethylene glycol terminated reactive end group suchas NHS or epoxide. Such polymers are commercially available from Sigmaand Shearwater polymers. Some other preferred polymers are PPO-PEO-PPOblock copolymers such as PLURONIC F68 terminated with NHS or SNHS group.PLURONIC or TETRONIC polymers are normally available with terminalhydroxyl groups. The hydroxyl groups are converted into acid group byreacting with succinic anhydride. The terminated acid groups are reactedwith N-hydroxysuccinimide in presence of DCC to generate NHS activatedPLURONIC polymer.

When Structures V-Y are functional polymers they may be multifunctionalgraft or branch type PEO or PEO block copolymers (TETRONICS) activatedwith terminal reactive groups such as NHS.

Structures U-Y in FIG. 5 need not have polymeric cores and may be smallmolecule crosslinkers. In that case, the core may comprise a smallmolecule like ethoxylated glycerol, tetraglycerol, hexaglycerol,inositol, trimethylolpropane, dilysine etc. to form the resultantcrosslinker.

Other variations of the core and the terminal nucleophilic functionalgroup in Structures U-Y in FIG. 5 may be employed, so long as theproperties of low tissue toxicity, water solubility, and reactivity withelectrophilic functional groups are maintained.

Preparation of Structures A-Y in FIGS. 1-5

The polymeric crosslinkers and functional polymers illustrated asStructures A-Y in FIGS. 1 to 5 may be prepared using variety ofsynthetic methods. Their preferred compositions are described in Table1.

TABLE 1 Preferred Crosslinkers and Functional Polymers Structure BriefDescription Typical Example A Water soluble, linear Polyethylene glycolor ethoxylated difunctional crosslinker or propylene glycol chainextended with functional polymer with water oligolactate and terminatedwith N- soluble core, extended with hydroxysuccinimide estersbiodegradable regions such as oligomers of hydroxyacids or peptidesequences which are cleavable by enzymes and terminated with proteinreactive functional groups B Water soluble, trifiunmcational Ethoxylatedglycerol chain extended crosslinker or functional with oligolactate andterminated with polymer with water soluble core, N-hydroxysuccinimideesters extended with biodegradable regions such as oligomers ofhydroxyacids or peptide sequences and terminated with protein reactivefunctional groups C Water soluble, tetrafunctional 4 arm polyethyleneglycol, erythritol crosslinker or functional or pentaerythritol orpentaerythritol polymer with water soluble core, chain extended witholigolactate and extended with biodegradable terminated with N- regionssuch as oligomers of hydroxysuccinimide esters hydroxyacids or peptidesequences and terminated with protein reactive functional groups D Watersoluble, tetrafunctional Ethoxylated ethylene diamine or crosslinker orfunctional polyethylene oxide-polypropylene polymer with water solublecore, oxide-polyethylene oxide block extended with biodegradablecopolymer like TETRONIC 908 regions such as oligomers of chain extendedwith hydroxyacids or peptide oligotrimethylene carbonate and sequencesand terminated with terminated with N- protein reactive functionalhydroxysuccinimide ester groups E Water soluble, branched Low molecularweight polyvinyl crosslinker or functional alcohol with 1% to 20%hydroxyl polymer with water soluble core, groups extended witholigolactate and extended with biodegradable terminated with N- regionssuch as oligomers of hydroxysuccinimide ester hydroxyacids or peptidesequences and terminated with protein reactive functional groups F Watersoluble, liner difunctional Polyethylene oxide-polypropylene crosslinkeror functional oxide-polyethylene oxide block polymer with water solublecore, copolymer surfactant like extended with biodegradable PLURONIC F68chain extended with regions such as oligomers of oligolactate andterminated with hydroxyacids or peptide amino acids such as lysine orpeptide sequences and terminated with sequences that may contain twoamines, carboxylic acid or thiols amine groups G Water soluble,trifunctional Ethoxylated glycerol chain extended crosslinker orfunctional with oligolactate and terminated with polymer with watersoluble core, aminoacid such as lysine extended with biodegradableregions such as oligomers of hydroxyacids or peptide sequences andterminated with amines, carboxylic acid or thiols H Water soluble,tetrafunctional 4 arm polyethylene glycol or tetra- crosslinker orfunctional erythritol chain extended with polymer with water solublecore, oligolactate and terminated with extended with biodegradableaminoacid such as lysine regions such as oligomers of hydroxyacids orpeptide sequences and terminated with amines, carboxylic acid or thiolsI Water soluble, tetrafunctional Ethoxylated ethylene diamine orcrosslinker or functional polyethylene oxide-polypropylene polymer withwater soluble core, oxide-polyethylene oxide block extended withbiodegradable copolymer like TETRONIC 908 regions such as oligomers ofchain extended with hydroxyacids or peptide oligotrimethylene carbonateand sequences and terminated with terminated with aminoacid such asamines, carboxylic acid or thiols lysine J Water soluble,multifunctional or Low molecular weight polyvinyl graft type crosslinkeror alcohol with 1-20% hydroxyl groups functional polymer with waterextended with oligolactate and soluble core, extended with terminatedwith aminoacid such as biodegradable regions such as lysine oligomers ofhydroxyacids or peptide sequences and terminated with amines, carboxylicacid or thiols K Water soluble, linear Difunctional oligolactic acidwith difunctional crosslinker or terminal carboxyl groups which arefunctional polymer such as activated with n- oligomers of hydroxyacidsor hydroxysulfosuccinimide ester or peptide sequences which areethoxylated n-hydroxysuccinimide terminated with protein reactive ester.functional groups L Water soluble branched Trifunctionaloligocaprolactone with trirunctional crosslinker or terminal carboxylgroups which are functional polymer such as activated with n- oligomersof hydroxyacids or hydroxysulfosuccinimide ester or peptide sequenceswhich are ethoxylated n-hydroxysuccinimide terminated with proteinreactive ester. functional groups M Water soluble, branchedTetrafunctional oligocaprolactone tetrafunctional crosslinker or withterminal carboxyl groups which functional polymer such as are activatedwith n- oligomers of hydroxyacids or hydroxysulfosuccinimide ester orpeptide sequences which are ethoxylated n-hydroxysuccinimide terminatedwith protein reactive ester. functional groups N Water soluble, branchedTetrafunctional oligocaprolactone tetrafunctional crosslinker or withterminal carboxyl groups which functional polymer such as are activatedwith n- oligomers of hydroxyacids or hydroxysulfosuccinimide ester orpeptide sequences which are ethoxylated n-hydroxysuccinimide terminatedwith protein reactive ester. functional groups O Water soluble, branchedMultifunctional oligolactic acid with multifunctional crosslinker orterminal carboxyl groups which are functional polymer such as activatedwith n- oligomers f hydroxyacids or hydroxysulfosuccinimide ester orpeptide sequences which are ethoxylated n-hydroxysuccinimide terminatedwith protein reactive ester. functional groups P Water soluble, linearPolyethylene glycol with terminal difunctional crosslinker or aminesgroups functional polymer terminated with amines, carboxylic acid orthiols functional groups Q Water soluble, branched Ethoxylated glycerolwith terminal trifunctional crosslinker or amines groups functionalpolymer terminated with amines, carboxylic acid or thiols as functionalgroup R Water soluble, branched 4 arm polyethylene glycol modifiedtetrafunctional crosslinker of to produce terminal amine groupsfunctional polymer terminated with amines, carboxylic acid or thiolsfunctional groups S Water soluble, branched Ethoxylated ethylene diamineor tetrafunctional crosslinker or polyethylene oxide-polyprophylenefunctional polymer terminated oxide-polyethylene oxide block withamines, carboxylic acid or copolymer like TETRONIC 908 thiols functionalgroups modified to generate terminal amine groups T Water soluble,branched or graft Polylysine, albumin, polyallyl amine crosslinker orfunctional polymer with terminal amines, carboxylic acid or thiolsfunctional groups U Water soluble, linear Polylysine, albumin, polyallylamine difunctional crosslinker or functional polymer terminated withprotein reactive functional groups V Water soluble branched Ethoxylatedglycerol terminated with trifunctional crosslinker orn-hydroxysuccinimide functional polymer terminated with protein reactivefunctional groups W Water soluble branched 4 arm polyethylene glycolterminated tetrafunctional crosslinker or with n-hydroxysuccinimideesters functional polymer terminated with protein reactive functionalgroups X Water soluble branched Ethoxylated ethylene diamine ortetrafunctional crosslinker or polyethylene oxide-polypropylenefunctional polymer terminated oxide-polyethylene oxide block withprotein reactive functional copolymer like TETRONIC 908 with groupsn-hydroxysuccinimide ester as end group Y Water soluble, branched orgraft Poly (vinyl pyrrolidinone)-co-poly polymer crosslinker or(n-hydroxysuccinimide acrylate) functional polymer with proteincopolymer (9:1), molecular weight < reactive functional groups 40000 Da

First, the biodegradable links of Structures A-J in FIGS. 1 and 2 may becomposed of specific di or multifunctional synthetic amino acidsequences which are recognized and cleaved by enzymes such ascollagenase, and may be synthesized using methods known to those skilledin the peptide synthesis art. For example, Structures A-E in FIG. 1 maybe obtained by first using carboxyl, amine or hydroxy terminatedpolyethylene glycol as a starting material for building a suitablepeptide sequence. The terminal end of the peptide sequence is convertedinto a carboxylic acid by reacting succinic anhydride with anappropriate amino acid. The acid group generated is converted to an NHSester by reaction with N-hydroxysuccinimide.

The functional polymers described in FIG. 2 may be prepared using avariety of synthetic methods. In a preferred embodiment, the polymershown as Structure F may be obtained by ring opening polymerization ofcyclic lactones or carbonates initiated by a dihydroxy compound such asPLURONIC F 68 in the presence of a suitable catalyst such as stannous2-ethylhexanoate. The molar equivalent ratio of caprolactone to PLURONICis kept below 10 to obtain a low molecular weight chain extensionproduct so as to maintain water solubility. The terminal hydroxyl groupsof the resultant copolymer are converted into amine or thiol by methodsknown in the art.

In a preferred method, the hydroxyl groups of a Pluronic-caprolactonecopolymer are activated using tresyl chloride. The activated groups arethen reacted with lysine to produce lysine terminatedPluronic-caprolactone copolymer. Alternatively, an amine-blocked lysinederivative is reacted with the hydroxyl groups of aPluronic-caprolactone copolymer and then the amine groups areregenerated using a suitable deblocking reaction.

Structures G, H, I and J in FIG. 2 may represent multifunctionalbranched or graft type copolymers having water soluble core extendedwith oligohydroxy acid polymer and terminated with amine or thiolgroups.

For example, in a preferred embodiment, the functional polymerillustrated as Structure G in FIG. 2 is obtained by ring openingpolymerization of cyclic lactones or carbonates initiated by atetrahydroxy compound such as 4 arm, tetrahydroxy polyethylene glycol(molecular weight 10,000 Da), in the presence of a suitable catalystsuch as stannous octoate. The molar equivalent ratio of cyclic lactoneor carbonate to PEG is kept below 10 to obtain a low molecular weightextension, and to maintain water solubility (polymers of cyclic lactonesgenerally are not as water soluble as PEG). Alternatively, hydroxyacidas a biodegradable link may be attached to the PEG chain usingblocking/deblocking chemistry known in the peptide synthesis art. Theterminal hydroxy groups of the resultant copolymer are activated using avariety of reactive groups known in the art. The CDI activationchemistry and sulfonyl chloride activation chemistry is shown in FIGS. 6and 7, respectively.

The most preferred reactive groups are N-hydroxysuccinimide esters,synthesized by any of several methods. In a preferred method, hydroxylgroups are converted to carboxylic groups by reacting them withanhydrides such as succinic anhydride in the presence of tertiary aminessuch as pyridine or triethylamine or dimethylaminopyridine (“DMAP”).Other anhydrides such as glutaric anhydride, phthalic anhydride, maleicanhydride and the like may also be used. The resultant terminal carboxylgroups are reacted with N-hydroxysuccinimide in the presence ofdicyclohexylcarbodiimide (“DCC”) to produce N-hydroxysuccinimide ester(referred as NHS activation). The NHS activation and crosslinkingreaction scheme is shown in FIG. 8. The most preferredN-hydroxysuccinimide esters are shown in FIG. 9.

In a preferred embodiment, the polymer shown as structure H is obtainedby ring opening polymerization of glycolide or trimethylene carbonateinitiated by a tetrahydroxy compound such as tetrafunctionalpolyethylene glycol (molecular weight 2000 Da) in the presence of acatalyst such as stannous 2-ethylhexoate. The molar equivalent ratio ofglycolide to PEG is kept from 2 to 10 to obtain a low molecular weightextension. The terminal hydroxy groups of the resultant copolymer areconverted into amine groups by reaction with lysine as mentionedpreviously. Similar embodiments can be obtained using analogous chainextension synthetic strategies to obtain structures F, G, I and J bystarting with the appropriate corresponding polyol.

Structures K, L, M, N and O in FIG. 3 are made using a variety ofsynthetic methods. In a preferred embodiment, the polymer shown asStructure L in FIG. 3 is obtained by ring opening polymerization ofcyclic lactones by a trihydroxy compound such as glycerol in thepresence of a catalyst such as stannous 2-ethylhexanoate. The molarequivalent ratio of cyclic lactone to glycerol is kept below 2, so thatonly low molecular weight oligomers are obtained. The low molecularweight oligomer ester is insoluble in water. The terminal hydroxy groupsof the resultant copolymer are activated using N-hydroxysulfosuccinimidegroups. This is achieved by converting hydroxy groups to carboxylicgroups by reacting with anhydrides such as succinic anhydride inpresence of tertiary amines. The resultant terminal carboxyl groups arereacted with N-hydroxysulfosuccinimide or N-hydroxyethoxylatedsuccinimide in the presence of dicyclohexylcarbodiimide (“DCC”) toproduce a sulfonated or ethoxylated NHS ester. The sulfonate or PEOchain on the succinimide ring gives water solubility to the oligoester.

The foregoing method generally is applied to solubilize only lowmolecular weight multi-branched oligoesters, with molecular weightsbelow 1000. In another variation of this method, various non-toxicpolyhydroxy compounds, preferably sugars, such as erythritol, xylitolare reacted with succinic anhydride in the presence of a tertiary amine.The terminal carboxyl group of succinated erythritol is esterified withN-hydroxysulfosuccinimide (FIG. 9). Similar embodiments may be obtainedusing analogous synthetic strategies to obtain structures K, and M-O bystarting with the appropriate starting materials.

Structures P-R may be synthesized by reacting the appropriate startingmaterial, such as a linear (P) or 2- or 3-arm branched PEG (Q, R) withhydroxy end groups, with lysine as mentioned previously, such that thearms of the PEG oligomers are capped with amine end groups. Structure Smay be synthesized, using a multistep reaction, from PEG, glycerol and adiisocyanate. In the first step a PEG diol is reacted with excessdiisocyanate, such as 4, 4′diphenyl methane diisocyanate (“MDI”),methylene-bis(4-cyclohexylisocyanate) (“HMDI”) orhexamethylenediisocyanate (“HDI”). After purification the resultant PEGdiisocyanate is added dropwise to excess glycerol or trimethylol propaneor other triol and reacted to completion. The purified product, nowhaving diol end groups, is again reacted with excess diisocyanate andpurified, yielding a PEG-tetra-isocyanate. This tetrafunctional PEGsubsequently may be reacted with excess PEG diols, yielding a 4 arm PEGsynthesized from a PEG diol oligomer. In the final step lysine endgroups are incorporated, as discussed previously.

Structure T may be synthesized as follows: First synthesize a randomcopolymer of PEG-monoacrylate and some other acrylate or combination ofacrylates, such that the final polyacrylate is water soluble. Otheracrylates include, but are not limited to , 2-hydroxyethylacrylate,acrylic acid, and acrylamide. Conditions may be varied to control themolecular weight as desired. In the final step, the acrylate is reactedwith lysine as discussed previously, using an appropriate quantity toachieve the desired degree of amination.

One method of synthesizing Structures U-Y is to usedicyclohexylcarbodiimide coupling to a carboxylate end group. ForStructures U-W, one can react the appropriate PEG-diol, -triol or-tetra-hydroxy starting material with excess succinic anhydride orglutaric anhydride such that all end groups are effectivelycarboxylated. Structures X and Y may be made in a manner similar to thatused for Structures S and T, except that in the last step instead of endcapping with lysine, end capping with succinic anhydride or glutaricanhydride is performed.

Preparation of Biocompatible Polymers

Several biocompatible crosslinked hydrogels may be produced using thecrosslinkers and functional polymers described in FIGS. 1 to 5.Preferred combinations of such polymers suitable for producing suchbiocompatible crosslinked polymers are described in Table 2. In Table 2,the crosslinker functional groups are N-hydroxy succinimide esters andthe functional polymer functional groups are primary amines.

TABLE 2 Biocompatible Polymers Synthesized from Crosslinkers andFunctional Polymers of Table 1 Functional Crosslinker Polymer StructureStructure Concentration Medium B or C H and R Molar Borate or triethanolEquivalent; >20% amine buffer, pH 7-10 W/V A, B or C H, P, Q, R MolarBorate or triethanol and S Equivalent; >20% amine buffer, pH 7-10 W/V YT, H, P and Molar Borate or triethanol Q Equivalent; >10% amine buffer,pH 7-10 W/V W, V H and J Molar Bicarbonate buffer, Equivalent; >20% pH7-10 W/V X I, J and H Molar Borate or triethanol Equivalent; >20% aminebuffer, pH 7-10 W/V

The reaction conditions for crosslinking will depend on the nature ofthe functional groups. Preferred reactions are conducted in bufferedaqueous solutions at pH 5 to 12. The preferred buffers are sodium boratebuffer (pH 10) and triethanol amine buffer (pH 7). Elevated pH increasesthe speed of electrophilic-nucleophilic reactions. In some embodiments,organic solvents such as ethanol or isopropanol may be added to improvethe reaction speed or to adjust the viscosity of a given formulation.

The synthetic crosslinked gels described above degrade due to hydrolysisof the biodegradable region. The degradation of gels containingsynthetic peptide sequences will depend on the specific enzyme and itsconcentration. In some cases, a specific enzyme may be added during thecrosslinking reaction to accelerate the degradation process.

When the crosslinker and functional polymers are synthetic (for example,when they are based on polyalkylene oxide), then it is desirable and insome cases essential to use molar equivalent quantities of thereactants. In some cases, molar excess crosslinker may be added tocompensate for side reactions such as reactions due to hydrolysis of thefunctional group.

When choosing the crosslinker and crosslinkable polymer, at least one ofpolymers must have more than 2 functional groups per molecule and atleast one degradable region, if it is desired that the resultantbiocompatible crosslinked polymer be biodegradable. For example, thedifunctional crosslinker shown as Structure A in FIG. 1 cannot form acrosslinked network with the difunctional polymers shown as Structure Fin FIG. 2 or Structure P in FIG. 4. Generally, it is preferred that eachbiocompatible crosslinked polymer precursor have more than 2 and morepreferably 4 or more functional groups.

Preferred electrophilic functional groups are NHS, SNHS and ENHS (FIG.9). Preferred nucleophilic functional groups are primary amines. Theadvantage of the NHS-amine reaction is that the reaction kinetics leadto quick gelation usually within 10 about minutes, more usually withinabout 1 minute and most usually within about 10 seconds. This fastgelation is preferred for in situ reactions on live tissue.

The NHS-amine crosslinking reaction leads to formation ofN-hydroxysuccinimide as a side product. The sulfonated or ethoxylatedforms of N-hydroxysuccinimide are preferred due to their increasedsolubility in water and hence their rapid clearance from the body. Thesulfonic acid salt on the succinimide ring does not alter the reactivityof NHS group with the primary amines.

The NHS-amine crosslinking reaction may be carried out in aqueoussolutions and in the presence of buffers. The preferred buffers arephosphate buffer (pH 5.0-7.5). triethanolamine buffer (pH 7.5-9.0) andborate buffer (pH 9.0-12) and sodium bicarbonate buffer (pH 9.0-10.0).

Aqueous solutions of NHS based crosslinkers and functional polymerspreferably are made just before the crosslinking reaction due toreaction of NHS groups with water. Longer “pot life” may be obtained bykeeping these solutions at lower pH (pH 4-5).

The crosslinking density of the resultant biocompatible crosslinkedpolymer is controlled by the overall molecular weight of the crosslinkerand functional polymer and the number of functional groups available permolecule. A lower molecular weight between crosslinks such as 600 willgive much higher crosslinking density as compared to a higher molecularweight such as 10,000. Higher molecular weight functional polymers arepreferred, preferably more than 3000 so as to obtain elastic gels.

The crosslinking density also may be controlled by the overall percentsolids of the crosslinker and functional polymer solutions. Increasingthe percent solids increases the probability that an electrophilicfunctional group will combine with a nucleophilic functional group priorto inactivation by hydrolysis. Yet another method to control crosslinkdensity is by adjusting the stoichiometry of nucleophilic functionalgroups to electrophilic functional groups. A one to one ratio leads tothe highest crosslink density.

Preparation of Biodegradable Polymers

The biodegradable crosslinkers described in FIGS. 1 and 3 may be reactedwith proteins, such as albumin, other serum proteins, or serumconcentrates to generate crosslinked polymeric networks. Briefly,aqueous solutions of the crosslinkers described in FIG. 1 and FIG. 3 (ata concentration of 50 to 300 mg/ml) are mixed with concentratedsolutions of albumin (600 mg/ml) to produce a crosslinked hydrogel. Thisreaction can be accelerated if a buffering agent, e.g., borate buffer ortriethanol amine, is added during the crosslinking step.

The resultant crosslinked hydrogel is a semisynthetic hydrogel whosedegradation depends on the degradable segment in the crosslinker as wellas degradation of albumin by enzymes. In the absence of any degradableenzymes, the crosslinked polymer will degrade solely by the hydrolysisof the biodegradable segment. If polyglycolate is used as thebiodegradable segment, the crosslinked polymer will degrade in 1-30 daysdepending on the crosslinking density of the network. Similarly, apolycaprolactone based crosslinked network will degrade in 1-8 months.The degradation time generally varies according to the type ofdegradable segment used, in the following order:polyglycolate<polylactate<polytrimethylene carbonate<polycaprolactone.Thus it is possible to construct a hydrogel with a desired degradationprofile, from a few days to months, using a proper degradable segment.

The hydrophobicity generated by biodegradable blocks such asoligohydroxy acid blocks or the hydrophobicity of PPO blocks in PLURONICor TETRONIC polymers are helpful in dissolving small organic drugmolecules. Other properties which will be affected by incorporation ofbiodegradable or hydrophobic blocks are: water absorption, mechanicalproperties and thermosensitivity.

Methods of Using Biocompatible Polymers

The biocompatible crosslinked polymers and their precursors describedabove may be used in a variety of applications, such as components oftissue adhesives, tissue sealants, drug delivery vehicles, woundcovering agents, barriers in preventing postoperative adhesions, andothers. These and other suitable applications are reviewed in Schlag andRedl, “Fibrin Sealant” in Operative Surgery, volumes 1-7 (1986), whichis incorporated herein by reference.

In Situ Formation

In many applications, the biocompatible crosslinked polymers of thisinvention typically will be formed “in situ” at a surgical site in thebody. The various methodologies and devices for performing “in situ”gelation, developed for other adhesive or sealant systems such fibringlue or sealant applications, may be used with the biocompatiblecrosslinked polymers of this invention. Thus, in one embodiment, anaqueous solution of a freshly prepared crosslinker (e.g.,SNHS-terminated oligolactide synthesized from a glycerol core inphosphate buffered saline (“PBS”) at pH 5 to 7.2) and a functionalpolymer (e.g., albumin or amine terminated tetrafunctional polyethyleneglycol at pH 10 in sodium borate) are applied and mixed on the tissueusing a double barrel syringe (one syringe for each solution). The twosolutions may be applied simultaneously or sequentially. In someembodiments, it is preferred to apply the precursor solutionssequentially so as to “prime” the tissue, resulting in improvedadherence of the biocompatible crosslinked polymer to the tissue. Wherethe tissue is primed, the crosslinker precursor is preferably applied tothe tissue first, followed by the functional polymer solution.

One may use specialized devices to apply the precursor solutions, suchas those described in U.S. Pat. Nos. 4,874,368; 4,631,055; 4,735,616;4,359,049; 4,978,336; 5,116,315; 4,902,281; 4,932,942; Published PatentCooperation Treaty Patent Application No. WO 91/09641; and R. A. Tange,“Fibrin Sealant” in Operative Medicine: Otolaryngology, volume 1 (1986),the disclosures of which are herein incorporated by reference.

Drug Delivery

The subject crosslinkers, functional polymer and their reactionproducts, the crosslinked materials advantageously may be used forlocalized drug therapy. Biologically active agents or drug compoundsthat may be added and delivered from the crosslinked polymer or gelinclude: proteins, glycosaminoglycans, carbohydrates, nucleic acid,inorganic and organic biologically active compounds where specificbiologically active agents include but are not limited to: enzymes,antibiotics, antineoplastic agents, local anesthetics, hormones,angiogenic agents, anti-angiogenic agents, growth factors, antibodies,neurotransmitters, psychoactive drugs, anticancer drugs,chemotherapeutic drugs, drugs affecting reproductive organs, genes, andoligonucleotides.

To prepare such crosslinked composition, the bioactive compoundsdescribed above are mixed with the crosslinkable polymer prior to makingthe aqueous solution or during the aseptic manufacturing of thefunctional polymer. This mixture then is mixed with the crosslinker toproduce a crosslinked material in which the biologically activesubstance is entrapped. Functional polymers made from inert polymerslike PLURONIC, TETRONICS or TWEEN surfactants are preferred in releasingsmall molecule hydrophobic drugs.

In a preferred embodiment, the active agent or agents are present in aseparate phase when crosslinker and crosslinkable polymers are reactedto produce a crosslinked polymer network or gel. This phase separationprevents participation of bioactive substance in the chemicalcrosslinking reaction such as reaction between NHS ester and aminegroup. The separate phase also helps to modulate the release kinetics ofactive agent from the crosslinked material or gel, where ‘separatephase’ could be oil (oil-in water emulsion), biodegradable vehicle, andthe like. Biodegradable vehicles in which the active agent may bepresent include: encapsulation vehicles, such as microparticles,microspheres, microbeads, micropellets, and the like, where the activeagent is encapsulated in a bioerodable or biodegradable polymers such aspolymers and copolymers of: poly(anhydride), poly(hydroxy acid)s,poly(lactone)s, poly(trimethylene carbonate), poly(glycolic acid),poly(lactic acid), poly(glycolic acid)-co-poly(glycolic acid),poly(orthocarbonate), poly(caprolactone), crosslinked biodegradablehydrogel networks like fibrin glue or fibrin sealant, caging andentrapping molecules, like cyclodextrin, molecular sieves and the like.Microspheres made from polymers and copolymers of poly(lactone)s andpoly(hydroxy acid) are particularly preferred as biodegradableencapsulation vehicles.

In using crosslinked materials which are described herein as drugdelivery vehicles, the active agent or encapsulated active agent may bepresent in solution or suspended form in crosslinker component orfunctional polymer solution component. The nucleophilic component,whether it be in the crosslinker or the functional polymer is thepreferred vehicle due to absence of reactive groups. The functionalpolymer along with bioactive agent, with or without encapsulatingvehicle, is administered to the host along with equivalent amount ofcrosslinker and aqueous buffers. The chemical reaction betweencrosslinker and the functional polymer solution readily takes place toform a crosslinked gel and acts as a depot for release of the activeagent to the host. Such methods of drug delivery find use in bothsystemic and local administration of an active agent.

In using the crosslinked composition for drug delivery as mentionedabove, the amount of crosslinkable polymer, crosslinker and the dosageagent introduced in the host will necessarily depend upon the particulardrug and the condition to be treated. Administration may be by anyconvenient means such as syringe, canula, trocar, catheter and the like.

Several methods for the formation of regional adhesion barriers aredescribed, in which any of a variety of water soluble macromericprecursors are used. The term “macromeric precursor” or “macromer” ismeant to connote an oligomeric or polymeric molecule that containsfunctional groups that enable further crosslinking. Preferably thefunctionality of a macromer molecule is >2 so that a crosslinked networkor hydrogel results upon crosslinking.

In one embodiment, a crosslinked regional barrier is formed in situ, forexample, by electrophilic-nucleophilic reaction, free radicalpolymerization initiated by a redox system or thermal initiation,wherein two components of an initiating system are simultaneously,sequentially or separately instilled in a body cavity to obtainwidespread dispersal and coating of all or most visceral organs withinthat cavity prior to gelation and crosslinking of the regional barrier.Once the barrier is formed, the organs remain isolated from each otherfor a predetermined period, depending upon the absorption profile of theadhesion barrier material.

Preferably, the barrier is selected to have a low stress at break intension or torsion, so as to not adversely affect normal physiologicalfunction of visceral organs within the region of application. Thebarrier also may contain a drug or other therapeutic agent.

Certain embodiments of the invention are accomplished by providingcompositions and methods to control the release of relatively lowmolecular weight therapeutic species using hydrogels. In accordance withthe principles of the present invention, a therapeutic species first isdispersed or dissolved within one or more relatively hydrophobic ratemodifying agents to form a mixture. The mixture may be formed intomicroparticles, which are then entrapped within a bioabsorbable hydrogelmatrix so as to release the water soluble therapeutic agents in acontrolled fashion. Alternatively, the microparticles may be formed insitu during crosslinking of the hydrogel.

In one method of the present invention, hydrogel microspheres are formedfrom polymerizable macromers or monomers by dispersion of apolymerizable phase in a second immiscible phase, wherein thepolymerizable phase contains at least one component required to initiatepolymerization that leads to crosslinking and the immiscible bulk phasecontains another component required to initiate crosslinking, along witha phase transfer agent. Pre-formed microparticles containing the watersoluble therapeutic agent may be dispersed in the polymerizable phase,or formed in situ, to form an emulsion. Polymerization and crosslinkingof the emulsion and the immiscible phase is initiated in a controlledfashion after dispersal of the polymerizable phase into appropriatelysized microspheres, thus entrapping the microparticles in the hydrogelmicrospheres. Visualization agents may be included, for instance, in themicrospheres, microparticles, and/or microdroplets.

Embodiments of the invention include compositions and methods forforming composite hydrogel-based matrices and microspheres havingentrapped therapeutic compounds. In one embodiment, a bioactive agent isentrapped in microparticles having a hydrophobic nature (herein called“hydrophobic microdomains”), to retard leakage of the entrapped agent.More preferably, the composite materials that have two phasedispersions, where both phases are absorbable, but are not miscible. Forexample, the continuous phase may be a hydrophilic network (such as ahydrogel, which may or may not be crosslinked) while the dispersed phasemay be hydrophobic (such as an oil, fat, fatty acid, wax, fluorocarbon,or other synthetic or natural water immiscible phase, genericallyreferred to herein as an “oil” or “hydrophobic” phase).

The oil phase entraps the drug and provides a barrier to release by slowpartitioning of the drug into the hydrogel. The hydrogel phase in turnprotects the oil from digestion by enzymes, such as lipases, and fromdissolution by naturally occurring lipids and surfactants. The latterare expected to have only limited penetration into the hydrogel, forexample, due to hydrophobicity, molecular weight, conformation,diffusion resistance, etc. In the case of a hydrophobic drug which haslimited solubility in the hydrogel matrix, the particulate form of thedrug may also serve as the release rate modifying agent.

Hydrophobic microdomains, by themselves, may be degraded or quicklycleared when administered in vivo, making it difficult to achieveprolonged release directly using microdroplets or microparticlescontaining the entrapped agent in vivo. In accordance with the presentinvention, however, the hydrophobic microdomains are sequestered in agel matrix. The gel matrix protects the hydrophobic microdomains fromrapid clearance, but does not impair the ability of the microdroplets ormicroparticles to release their contents slowly. Visualization agentsmay be included, for instance, in the gel matrix or the microdomains.

In one embodiment, a microemulsion of a hydrophobic phase and an aqueoussolution of a water soluble molecular compound, such as a protein,peptide or other water soluble chemical is prepared. The emulsion is ofthe “water-in-oil” type (with oil as the continuous phase) as opposed toan “oil-in-water” system (where water is the continuous phase). Otheraspects of drug delivery are found in commonly assigned U.S. patentapplication Ser. Nos. 09/134,287 entitled “Composite Hydrogel DrugDelivery Systems”; Ser. No. 09/390,046 entitled “Methods and Apparatusfor Intraluminal Deposition of Hydrogels”; and Ser. No. 09/134,748entitled “Methods for Forming Regional Tissue Adherent Barriers and DrugDelivery Systems”, each of which are hereby incorporated by reference.

In another aspect of the present invention, the hydrogel microspheresare formed having a size that will provide selective deposition of themicrospheres, or may linked with ligands that target specific regions orotherwise affect deposition of the microspheres within a patient's body.

Controlled rates of drug delivery also may be obtained with the systemof the present invention by degradable, covalent attachment of thebioactive molecules to the crosslinked hydrogel network. The nature ofthe covalent attachment can be controlled to enable control of therelease rate from hours to weeks or longer. By using a composite madefrom linkages with a range of hydrolysis times, a controlled releaseprofile may be extended for longer durations.

Composite Biomaterials

The biocompatible crosslinked polymers of this invention optionally maybe reinforced with flexible or rigid fibers, fiber mesh, fiber cloth andthe like. The insertion of fibers improves mechanical properties likeflexibility, strength, and tear resistance. In implantable medicalapplications, biodegradable fibers, cloth, or sheets made from oxidizedcellulose or poly(hydroxy acid)s polymers like polylactic acid orpolyglycolic acid, are preferred. Such reinforced structures may beproduced using any convenient protocol known in the art.

In a preferred method, aqueous solutions of functional polymers andcrosslinkers are mixed in appropriate buffers and proportions are addedto a fiber cloth or net such as INTERCEED (Ethicon Inc., New Brunswick,N.J.). The liquid mixture flows into the interstices of the cloth andbecomes crosslinked to produce a composite hydrogel. Care is taken toensure that the fibers or fiber mesh are buried completely inside thecrosslinked hydrogel material. The composite structure can be washed toremove side products such as N-hydroxysuccinimide. The fibers used arepreferably hydrophilic in nature to ensure complete wetting of thefibers by the aqueous gelling composition.

EXAMPLES

The following non-limiting examples are intended to illustrate thesynthesis of new biocompatible crosslinked polymers and theirprecursors, and their use in making several medical products. Thoseskilled in the art will appreciate that modifications can be made tothese examples, drawings, illustrations and claims that are intended tofall within the scope of the present invention.

Materials and Equipment

Polyethylene glycol was purchased from various sources such asShearwater Polymers, Union Carbide, Fluka and Polysciences.Multifunctional hydroxyl and amine terminated polyethylene glycol werepurchased from Shearwater Polymers, Dow Chemicals and Texaco. PLURONICand TETRONIC series polyols were purchased from BASF Corporation.DL-lactide, glycolide, caprolactone and trimethylene carbonate wasobtained from commercial sources like Purac, DuPont, Polysciences,Aldrich, Fluka, Medisorb, Wako and Boehringer Ingelheim.N-hydroxysulfosuccinimide was purchased from Pierce. All other reagents,solvents were of reagent grade and were purchased from commercialsources such as Polysciences, Fluka, Aldrich and Sigma. Most of thereagents and solvents were purified and dried using standard laboratoryprocedures such as described in D. D. Perrin et al., Purification ofLaboratory Chemicals (Pergamon Press 1980).

General Analysis

The polymers synthesized according to these examples were chemicallyanalyzed using structure-determining methods such as nuclear (proton andcarbon-13) magnetic resonance spectroscopy, infrared spectroscopy.Molecular weights were determined using high pressure liquidchromatography and gel permeation chromatography. Thermalcharacterization of the polymers, including melting point and glasstransition temperatures, were performed using differential scanningcalorimetric analysis. Aqueous solution properties such as micelle andgel formation was determined using fluorescence spectroscopy, UV-visiblespectroscopy and laser light scattering instruments.

In vitro degradation of the polymers was followed gravimetrically at 37°C., in an aqueous buffered medium such as phosphate buffered saline (atpH 7.2). In vivo biocompatibility and degradation life times wasassessed by injecting or forming a gelling formulation directly into theperitoneal cavity of a rat or rabbit and observing its degradation overa period of 2 days to 12 months.

Alternatively, the degradation was also assessed by prefabricating asterile implant, made by a process like solution casting, thensurgically implanting the implant within an animal body. The degradationof the implant over time was monitored gravimetrically or by chemicalanalysis. The biocompatibility of the implant was assessed by standardhistological techniques.

Example 1 Synthesis of a Water-Soluble Difunctional, BiodegradableFunctional Polymer Based on Polyalkylene Oxide Block Copolymer

First, Polyethylene glycol-co-polycaprolactone polyol (“F68C2”) wassynthesized as follows:

30 g of PLURONIC F68 was dried under vacuum at 110° C. for 6 h and thenmixed with 1.710 g of caprolactone and 30 mg of stannous2-ethylhexanoate in a glass sealing tube. The glass tube then was sealedunder nitrogen atmosphere and heated to 170° C. and maintained at thistemperature for 16 h. The PLURONIC F68-caprolactone polymer was cooledand recovered by breaking the glass sealing tube, and then furtherpurified by several precipitations from a toluene-hexanesolvent-nonsolvent system.

The polymer then was dried in vacuum at 40° C. and used immediately inthe activation reaction described below:

Reaction with succinic anhydride (“11F68C2S”):

30 g of PLURONIC F68-caprolactone copolymer was dissolved in 200 ml dryN,N-dimethyl formamide (“DMF”) and 0.845 g of succinic anhydride wasadded to the reaction mixture. The mixture was heated to 100° C. under anitrogen atmosphere for 16 h. The solution then was cooled and added to4000 ml hexane to precipitate the carboxyl terminated polymer. It wasfurther purified by repeated (3 times) precipitation from atoluene-hexane solvent-nonsolvent system. The polymer was dried undervacuum at 40° C.

This polymer was immediately used in activation reaction describedbelow:

Activation of Carboxyl Groups with N-Hydroxysuccinimide (“F68C2SSNHS”):

30 g of PLURONIC F68-caprolactone succinate copolymer was dissolved in200 ml dry DMF. The solution was cooled to 4° C. and 1.504 g of1,3-dicyclohexylcarbodiimide (“DCC”) and 1.583 g ofN-hydroxysulfosuccinimide (“SNHS”) were added to the reaction mixture.The mixture was stirred at 4° C. for 6 h and then stirred overnight atroom temperature under nitrogen atmosphere. Dicyclohexylurea was removedby filtration and the F68C2S-SNHS derivative was isolated by removingthe DMF under vacuum and repeated precipitation using a toluene-hexanesolvent-nonsolvent system. The product was stored under nitrogenatmosphere at −20° C.

Example 2 Amine Terminated Synthetic Biodegradable Crosslinkable Polymer

Reaction of F68TMC2SSNHS with Lysine.

3.55 g of lysine was dissolved in 200 ml 0.1 M borate buffer (pH 8.5).The mixture was cooled to 0° C. in ice bath and 10 g of F68C2SSNHS wereadded to the mixture. The mixture was stirred for 6 h at roomtemperature and lyophilized. The lyophilized powder was dissolved in 30ml toluene and filtered. The filtrate was added to 4000 ml cold diethylether. The precipitated amine terminated polymer was recovered byfiltration and dried under vacuum. The polymer was stored under argon at−20° C.

Example 3 Synthesis of Carboxyl Terminated Oligolactic Acid PolymerActivated with N-hydroxysulfosuccinimide

Synthesis of difunctional oligolactate with terminal carboxyl acidend-groups activated with N-hydroxysulfosuccinimide groups.

Part 1: Synthesis of Oligomeric Poly(Lactic Acid) with Terminal CarboxylAcid Groups (“PLA-S”).

In a 250 ml 3 neck flask equipped with mechanical stirrer, nitrogeninlet and distillation condenser, 2 grams of succinic acid and 34.1 ml 1N HC 1 and 3.83 g L-lactic acid, sodium salt were charged. The flask wasthen immersed in a silicone oil bath maintained at 150° C. Most of thewater from the reaction mixture was removed over period of 5 hours bydistillation. The remaining water was removed by heating the reactionmixture under vacuum at 180° C. for 15 h. The reaction mixture wascooled and lyophilized at 0° C. to remove traces of water. The productwas isolated by dissolving in toluene and precipitating in hexane. Theprecipitated polymer was isolated by filtration and dried in vacuum for48 h at 60° C.

Part 2: Activation of Terminal Groups with N-HydroxysulfosuccinimideGroup:

A 3 necked flask equipped with magnetic stirrer and nitrogen inlet wascharged with 2 g of PLA-S copolymer and 20 ml DMF. The solution wascooled 4° C. and 3.657 g of N-hydroxysulfosuccinimide and 3.657 g of1,3-dicyclohexyl carbodiimide were added to the reaction mixture. Themixture was stirred at 4° C. for 6 h and overnight at room temperatureunder nitrogen atmosphere. Dicyclohexylurea was removed by filtrationand SNHS derivative was by isolated by removing the DMF under vacuum andrepeated precipitation using toluene-hexane solvent-nonsolvent system.The product was stored under nitrogen atmosphere at 4° C.

Example 4 Preparation of Polyethylene Glycol Based TetrafunctionalCrosslinker

Part 1: Synthesis of Tetrafunctional PolyethyleneGlycol-co-polyglycolate Copolymer (“4PEG2KG”):

30 grams of 4 arm polyethylene glycol, molecular weight 2000 (“4PEG2K”)was dried at 100° C. for 16 hours prior to use. 30 grams 4PEG2K. 7.66 gof glycolide and 25 mg of stannous 2-ethylhexanoate were charged into a3 necked flask equipped with a Teflon coated magnetic stirring needle.The flask was then immersed into silicone oil bath maintained at 160° C.The polymerization reaction was carried out for 16 h under nitrogenatmosphere. At the end of the reaction, the reaction mixture wasdissolved in 100 ml toluene. The hydroxy terminated glycolate copolymerwas isolated by pouring the toluene solution in 4000 ml cold hexane. Itwas further purified by repeated dissolution-precipitation process fromtoluene-hexane solvent-nonsolvent system and dried under vacuum at 60°C. It then was immediately used for end capping reaction mentionedbelow:

Part 2: Conversion of Hydroxyl Groups into Carboxylic Groups(“4PEG2KGS”) and SNHS Ester.

30 g of 4PEG2KG copolymer was dissolved in 150 ml dry pyridine. 8.72 gof succinic anhydride was added to it and the solution was refluxed for2 h under nitrogen atmosphere. The polymer was isolated by pouring thecold pyridine solution to 4000 ml hexane. The acid terminated polymer(“4PEG2KGS”) was used in SNHS activation reaction. Briefly, to asolution of 30 g of 4PEG2KGS in 300 ml dry methylene chloride were added10.58 g of SNHS and 10.05 g DCC. The reaction mixture was stirredovernight under nitrogen atmosphere. Dicyclohexylurea was removed byfiltration. The filtrate was evaporated and the residue obtained wasredissolved in 100 ml toluene. The toluene solution was precipitated in2000 ml hexane. The SNHS activated polymer was stored under nitrogenatmosphere until further use.

Example 5 Sulfonyl Chloride Activated Crosslinkers

Activation of tetrafunctional polyethylene glycol-co-polyglycolatecopolymer (“4PEG2KGS”) with tresyl chloride.

30 g of 4PEG2KG was dissolved in 10 ml dry benzene. The solution wascooled to 0° C. and 5.92 g of triethyl amine and 10.70 g tresyl chloridewere added under nitrogen atmosphere. After refluxing for 3 h undernitrogen atmosphere, the reaction mixture was cooled and filtered toremove triethylamine hydrochloride. The filtrate was poured into 3000 mlhexane to precipitate the activated polymer. The residue was redissolvedin THF and filtered over neutral alumina to remove traces oftriethylamine hydrochloride. The polymer was recovered by adding the THFsolution to 3000 ml diethyl ether and stored under nitrogen atmosphere.

Example 6 Synthesis of Multifunctional Oligopolycaprolactone Terminatedwith SNHS

Part 1: Synthesis of Polycaprolactone (“PCL1”).

2.00 g of glycerol, 8.17 g of caprolactone and 50 mg of stannous2-ethylhexanoate were charged into 100 ml Pyrex pressure sealing tube.The tube was frozen in liquid nitrogen and connected to vacuum line for10 minutes. The tube then was connected to argon gas line and sealedunder argon. The sealed reaction mixture then was immersed in oil bathmaintained at 160° C. and polymerization was carried out for 16 h at160° C. The polymer was recovered by dissolving it in 30 ml toluene andprecipitating in 2000 ml cold hexane. The precipitated liquid oligomerwas recovered and dried under vacuum for 1 day at 60° C.

Part 2: End-Capping of PCL1 with Succinic Anhydride (“PCL-S”):

10 g of PCL1 was dissolved in 150 ml dry benzene. About 50 ml of benzenewas distilled to remove traces of water from the reaction mixture. Thesolution was cooled to 30° C. To this warm solution, 6.67 g of triethylamine and 7.86 g of succinic anhydride were added. The reaction mixturewas then refluxed for 6 h and concentrated by distillation under vacuum.The product was recovered by adding the filtrate to 2000 ml cold dryhexane.

Part 3: Activation of PCL-S with SNHS:

PCL1-succinate (5.0 g) was dissolved in 10 ml of anhydrous methylenechloride, cooled to 0° C. and 7.82 g of N-hydroxysulfosuccinimide and7.42 N, N-dicyclohexylcarbodiimide were added under stirring. Afterstirring the mixture overnight, the precipitated dicyclohexylurea wasremoved by filtration and the solution was concentrated by removingsolvent. The ¹H-NMR spectrum showed succinimide singlet at 2.80 ppm(2H).

Example 7 Preparation of Polyethylene Glycol-co-polytrimethyleneCarbonate Copolymer Terminated with N-hydroxysuccinimide

Preparation of tetrafunctional polyethylene glycol-co-polytrimethylenecarbonate copolymer (“4PEG10KTMC2”).

30 g of tetrahydroxy polyethylene glycol, molecular weight 10000, wasdried under vacuum at 90-100° C. in a glass sealing tube. The tube thenwas cooled and transferred inside an air bag where 2.45 g oftrimethylene carbonate and 20 mg of stannous octoate were added to thetube. The glass tube was then sealed under vacuum and heated withstirring at 155° C. and maintained at this temperature for 16 h. Thepolyethylene glycol-co-polytrimethylene carbonate polymer was cooled andrecovered by breaking the glass sealing tube. It was further purified byseveral precipitations from toluene-hexane solvent-nonsolvent system.

Part 2: Synthesis of Glutarate Derivative of 4PEG10KTMC2(“4PEG10KTMC2G”):

10 g of 4PEG10KTMC was dissolved in 120 ml dry toluene. About 50 ml oftoluene was distilled to remove traces of water from the reactionmixture. The warm solution was cooled to 60° C. To this solution, 1.23 gof triethyl amine and 1.40 g of glutaric anhydride were added. Thereaction mixture was heated to 60° C. for 1 h and filtered. The productwas recovered by adding the filtrate to 2000 ml cold dry hexane.

Part 3: Activation of Terminal Carboxyl Groups UsingN-hydroxysuccinimide (“4PEG10KTMC2GNHS”):

30 g of 4PEG10KTMC2G was dissolved in 100 ml of dry DMF and 1.53 g ofN-hydroxysuccinimide and 5 g molecular sieves 3 Å were added. 1.28 g ofDCC dissolved in 5 ml dry DMF was added dropwise and the reactionmixture was kept at room temperature for 24 h under nitrogen atmosphere.The mixture was diluted with 50 ml cold benzene and precipitated usingcold hexane. The precipitate was collected on a sintered glass filterwith suction. The dissolution and precipitation procedure was thenrepeated three times, using toluene-diethyl ether as solvent-nonsolventsystem and dried under vacuum. The product was stored under nitrogenatmosphere at −20° C. until further use.

Example 8 Succinated Polyhydroxy Compounds Activated withN-hydroxysulfosuccinimide ES

10 g of erythritol was dissolved in 200 ml dry toluene. About 50 ml oftoluene was distilled to remove traces of water from the erythritol. Thesolution was cooled to 50-60° C. and 20 ml pyridine and 8.58 g ofsuccinic anhydride were added to the solution. The reaction mixture wasthen refluxed for 3 h and unreacted pyridine and toluene were evaporatedto dryness under reduced pressure. The residue was used in activationreaction.

Part 2: Activation of ES with SNHS:

Erythritol-succinate (ES, 2.0 g) was dissolved in 10 ml of anhydrousdimethyl formamide (“DMF”), cooled to 0° C. and 3.47 g ofN-hydroxysulfosuccinimide and 3.30 N, N-dicyclohexylcarbodiimide wereadded under stirring. After stirring the mixture overnight, theprecipitated dicyclohexylurea was removed by filtration and the solutionwas concentrated by removing solvent. It was further purified by columnchromatography.

Example 9 Preparation of Synthetic Crosslinked Biodegradable Gels

1.57 g (0.8 mM) of 4 arm amine terminated polyethylene glycol molecularweight 2000 was dissolved in 10 ml 0.1 M sodium borate buffer at pH 9.52 g of 4 arm SNHS activated 4PEG2KGS polymer (molecular weight 2500) wasdissolved in phosphate buffered saline. These two solutions were mixedto produce a crosslinked gel. In another variation of this method, the4PEG2KGS polymer solid was directly added to the amine terminatedpolymer solution to produce a crosslinked polymer.

In another variation, a crosslinker consisting of an equimolar solutionof dilysine can be used in place of the 4 arm PEG amine solution to forma hydrogel. Gelation was seen to occur within 10 seconds of mixing thetwo solutions. Similarly, other crosslinkers described in examples 1 to7 may be reacted in molar equivalent proportions with other amineterminated polymers such as albumin or amine terminated biodegradablepolymers similar to described in Example 2. The preferred compositionsfor making biodegradable hydrogels were described in Table 2. The amineterminated polymer solution described above was added with 0.1% of F Dand C blue or indigo dye prior to crosslinking reaction. The addition ofdye allows the preparation of colored gels.

Example 10 Preparation of Composite Synthetic Crosslinked ColoredBiodegradable Gels

3 grams of bovine serum albumin was dissolved in 3 ml of phosphatebuffered solution. Commercial sutures based on synthetic biodegradablepolymers, such as Vicryl was cut/ground into several small pieces (sizeless than 1 mm) using cryogenic grinding. These colored suture particles(approximately 100 mg) were mixed with the albumin solution to form asuspension. 100 mg of crosslinker such as 4PEG10KTMC2GNHS was mixed with0.2 ml of albumin suspension. This viscous solution then was mixed with40 mg of triethanol amine (buffering agent). The addition of triethanolamine gels the solution in 60 seconds. The colored suture particlesentrapped in the crosslinked gel help to visualize the gel especiallywhen under laparoscopic conditions and also acts to strengthen thehydrogel as a reinforcing agent. The suture particles in above examplescan be replaced with biodegradable microparticles loaded with drugs orbioactive compounds.

Example 11 Formulation of SG-PEG with Di-lysine

A four arm PEG with SG end groups (Shearwater Polymers, approx. 9,100g/mol, 0.704 grams, 6.5×10⁻⁵ moles) was dissolved in 2.96 g 0.01 M pH4.0 phosphate buffer (19.2% solids). Di-lysine (Sigma, 347.3 g/mol, 0.03grams, 8.7×10⁻⁵ moles) was dissolved in 3.64 grams of 0.1 M pH 9.5borate buffer (0.8% solids). On combination of the two solutions, thepercent solids was 10%. The di-lysine has 3 amine groups. The SG-PEG has4 NHS groups. After correction for the less than 100% degree ofsubstitution on the SG-PEG, the formulation gives a 1:1 stoichiometry ofamine groups to NHS groups.

Example 12 Formulation of SG-PEG with Tri-lysine

A four arm PEG with SG end groups (Shearwater Polymers, approx. 9,100g/mol, 0.675 grams, 6.2×10⁻⁵ moles) was dissolved in 2.82 g 0.01 M pH4.0 phosphate buffer (19.3% solids). Tri-lysine (Sigma, 402.5 g/mol,0.025 grams, 6.2×10⁻⁵ moles) was dissolved in 3.47 grams of 0.1 M pH 9.5borate buffer (0.7% solids). On combination of the two solutions, thepercent solids was 10%. The tri-lysine has 4 amine groups. The SG-PEGhas 4 NHS groups. After correction for the less than 100% degree ofsubstitution on the SG-PEG, the formulation gives a 1:1 stoichiometry ofamine groups to NHS groups.

Example 13 Formulation of SG-PEG with Tetra-lysine

A four arm PEG with SG end groups (Shearwater Polymers, approx. 9,100g/mol, 0.640 grams, 5.9×10⁻⁵ moles) was dissolved in 2.68 g 0.01 M pH4.0 phosphate buffer (19.2% solids). Tetra-lysine (Sigma, 530.7 g/mol,0.025 grams, 4.7×10-′ moles) was dissolved in 3.30 grams of 0.1 M pH 9.5borate buffer (0.8% solids). On combination of the two solutions, thepercent solids was 10%. The tetra-lysine has 5 amine groups. The SG-PEGhas 4 NHS groups. After correction for the less than 100% degree ofsubstitution on the SG-PEG, the formulation gives a 1:1 stoichiometry ofamine groups to NHS groups.

Example 14 Gel Time Measurement

The amine solution (100 μL) was aliquotted into a 100×13 test tube. Aflea-stirbar (7×2 mm, Fisher Scientific p/n 58948-976) was placed in thetest tube. The test tube was held stationary over a digital magneticstirrer (VWR Series 400S Stirrer) set at 300 rpm. A 1 cc tuberculinsyringe (Becton Dickinson, p/n BD309602) was filled with 100 μL of theester solution. The syringe was inserted up to the flanges so that thedistal end was just over the amine solution. Simultaneously the plungerwas depressed and a stop watch started. When the solution solidifiessufficiently so that the stir bar stops spinning, the stop watch wasstopped. Each solution was measured in triplicate and the mean ±1standard deviation was plotted. Results for the formulations of examples1, 2 and 3 are shown in FIG. 11.

Example 15 Change in Gel Time as a Function of Ester Solution Age

An important characteristic of these systems is the loss in reactivityover time from reconstitution of the ester solution. This loss inreactivity occurs due to hydrolysis of the N-hydroxysuccinimidyl ester,before the activated molecule can combine with its respectivenucleophilic functional group. The loss of reactivity was characterizedby measuring the change in gel time as a function of time fromreconstitution of the NHS ester solution. The gel time was measuredperiodically. The NHS ester solution was stored at ambient conditionsduring this measurement. Results for the solutions described in Examples11, 12 and 13 are shown in FIG. 12.

Example 16 Gel Formation at Different Percent Solids from 4 ArmCM-HBA-NS PEG and Lys-Lys

Using the gel time method described in Example 13, five different gelcompositions were made using carboxymethylhydroxybutyrate-hydroxysuccinimide end-capped 4 arm PEG (CM-HBA)(Shearwater Polymers) and di-lysine (Sigma). The formulations are listedbelow in Table 3.

TABLE 3 Phosphate Lys-Lys Borate Conc. (%) CM-HBA (g) (g) (g) (g) 8.50.2469 1.264 0.01 1.5012 10 0.2904 1.2209 0.012 1.4994 12.5 0.363 1.14830.015 1.4964 15 0.4356 1.0757 0.018 1.4936 20 0.5808 0.9305 0.024 1.4876

The formulations were adjusted to give a 1 to 1 ratio of electrophilicfunctional end groups on the CM-HBA (4) to nucleophilic reactive groupson the di-lysine (“Lys-Lys”)(3). The CM-HBA quantities were dissolved in0.01 M pH 5.0 phosphate buffer. The di-lysine was dissolved in 0.1 M pH11 borate buffer. Gel time results are shown in FIG. 13. This data alsoshows that the higher percent solids solutions also are the most stablewith respect to retention of speed of reaction.

Example 17 Degradation of Hydrogels

Hydrogel plugs made during the gel time measurements of Example 14 wereplaced in approximately 25 mL 0.1 M phosphate buffered saline at pH 7.4in 50 mL Falcon tubes and placed in a constant temperature bath at 37°C. The hydrogel plugs were observed visually at periodic intervals andthe time of gel disappearance noted. The data are plotted in FIG. 14.

Example 18 Precursor Spray Procedure to form a 7.5% Solids Hydrogel from4 Arm SG and Dilysine

An ethylene oxide sterilized air assisted sprayer was used inconjunction with aqueous solutions of polymerizable monomers. Solution 1consisted of a 14.4% solution of 4 arm SG (MW 10,000 purchased fromShearwater Polymers) dissolved in 0.01 M phosphate buffer at pH 4.0 andwas sterile filtered (Pall Gelman syringe filter, p/n 4905) and drawn upin a sterile 5 cc syringe. Solution 2 consisted of a 1.2% solution of adilysine (purchased from Sigma Chemicals) dissolved in 0.1 M boratebuffer at pH 11 with 0.5 mg/mL methylene blue for visualization and wasalso sterile filtered and drawn up in a sterile 5 cc syringe. Thesesolutions, when combined 1:1 on a volumetric basis, resulted in a 1:1ratio of NHS ester to amine end group. The final % solids aftercombination was 7.5%. The two syringes were individually loaded in thetwo separate receptacles through a luer-lok type of linkage. Airflowfrom a regulated source of compressed air (an air compressor such asthose commercially available for airbrushes) was connected to the deviceusing a piece of Tygon tube. On compressing the syringe plungers asteady spray of the two liquid components was observed. When this spraywas directed to a piece of tissue (rat cecum) a hydrogel coating wasobserved to form on the surface of the tissue. This hydrogel coating wasrinsed with saline (the hydrogel coating is resistant to rinsing) andwas observed to be well adherent to the tissue surface. Within a shortperiod of time (less than a minute) an area of 10 cm×5 cm could becoated with ease.

Example 19 Precursor Spray Procedure to form a 12.5% Solids Hydrogelfrom 4 Arm CM and Dilysine

A hydrogel barrier film made from 4 arm CM-HBA NS (MW 10,000 purchasedfrom Shearwater Polymers), and dilysine was similarly prepared andsprayed as described in Example 18. In the present example the 4 arm CMsolution was made up to 24.0% solids and the dilysine solution was madeup to 1.0% solids such that on combination in an equal volume deliverysystem a 1:1 ratio of NHS to amine end groups results, giving a final %solids of 12.5%.

Example 20 Spray Application of Crosslinker and Polymer to fromCrosslinked Film

Two solutions (component A and component B) were prepared. Component Aconsisted of dilysine in 0.1 M borate buffer, pH 9.5. Component Bconsisted of either 4 arm SG-PEG or 4 arm CM-HBA-NS in 0.01 M phosphatebuffer, pH 4.0 These solutions were prepared such that the amine toester stoichiometric ratio was 1:1 and the final total solutionconcentration was 7.5% or 12.5%, respectively.

A FIBRIJECT™ (Micromedics, Inc.) 5 cc syringe holder and cap was used,preloaded with 5 cc of each solution and attached to a dual barrelatomizing sprayer. The sprayer has two hubs for the syringes to connectto allowing the two fluids to be advanced through two separate lumensover any preset distance. A third hub exists for the application of theatomizing gas. Air was used in this example. The distal tip of thesprayer contains a chamber where the gas expands out of an introductiontube, then flows past the two polymer solution nozzles in an annularspace around each. The gas is accelerated in the annular spaces using aflow rate suitable for the complete atomization of the two fluid streams(˜2 L/min.). Two overlapping spray cones are thus formed allowing forwell mixed, thin, uniform coatings to be applied to surfaces.

Example 21 Adhesion Prevention in Rat Cecum Model

Surgical Procedure

Male Sprague Dawley rats (250-300 grams,) were anesthetized with anintramuscular 4 ml/kg “cocktail” of KETAMINE (25 mg/ml), XYLAZINE (1.3mg/mL) and ACEPROMAZINE (0.33 mg/mL). The abdominal area was shaved andprepped for aseptic surgery. A midline incision was made to expose theabdominal contents. The cecum was identified and location within theabdomen was noted. The cecum was pulled out of the abdomen and thesurface of one side was abraded using dry sterile gauze. A technique ofabrading one area by stroking the surface 12 times with the gauze wasused. The cecal arterial supply was interrupted using bipolarcoagulation along the entire surface area of the damaged cecum.

The opposing abdominal sidewall which lays in proximity to the damagedcecal surface was deperitonealized with a scalpel blade and theunderlying muscle layer was scraped to the point of hemorrhaging.

The cecum was sprayed with either the SG-PEG system or the CM-HBA-NSsystem using the air assisted spray method described in the precedingexample. The cecum was placed with the damaged (ischemic area) side upopposite the damaged side wall. Active bleeding was controlled beforeclosing. The peritoneum and muscle wall was closed with 3-0 nylon andthe skin was closed with 4-0 silk. Rats were returned to their cages forone to two weeks at which time evaluation of the adhesion between theside wall and cecum was noted. The rats were killed at 10 days and thetenacity and extent of adhesion was evaluated. The results aresummarized in Table 4.

TABLE 4 Rat Material Reference # Applied Example Finding on Day 10 4037.5% 4aSG Example 18 Small amount of gel present with Lys-Lys on cecum.No adhesions from w/MB cecum to sidewall. No gel on sidewall 404 7.5%4aSG Example 18 Some mesentery stuck to cecum. with Lys-Lys No gel. Noadhesions. w/MB 405 7.5% 4aSG Example 18 Small amount of gel presentwith Lys-Lys on cecum. Some mesentery w/MB stuck to cecum and sidewall.Some gel between mesentery and cecum where stuck. No adhesions. 40612.5% 4aCM Example 19 No gel present. No adhesions. with Lys-Lys w/MB407 12.5% 4aCM Example 19 No gel on cecum or sidewall. with Lys-Lys Noadhesions. w/MB 408 12.5% 4aCM Example 19 Rat died post-op (anesthesiawith Lys-Lys overdose). w/MB

Example 22

This example is directed to concentrations of coloring agent for use inan in situ crosslinked hydrogel coating. A 140 mg amount of four-armprimary amine terminated polyethylene glycol molecule with a molecularweight of approximately 10,000 was dissolved in sodium borate buffer pH9.5. An 84 mg amount of four arm NHS activated polyethylene glycolpolymer (SPA-3400, Shearwater Corp., Huntsville, Al, molecular weightapproximately 3400) was dissolved in pH 5.0 acetate buffer. MethyleneBlue was added to the borate buffered solutions at concentrations of0.1, 0.5, and 1.0 mg/ml.

A standard laparoscopic sprayer was used in a laparoscopic trainer tospray the surfaces of pieces of lunch meat with an approximately 1:1mixture of the solutions. The mixture formed a gel in about 3-6 secondson the surfaces. The sprayed gel was observed through a 10 mmlaparoscope and videotaped. The tapes were reviewed to assess the effectof the coloring agent. The 0.5 mg/ml and 1.0 mg/ml solutions of coloringagent created a gel that was readily observable and similar invisibility. The 0.1 mg/ml solution of coloring agent created a gel thatwas light in color and more difficult to observe compared to the othersolutions. Many previous experiments had already shown that gels with nocoloring agents were very difficult to observe visually. Controlexperiments performed without the presence of methylene blue showed thatthe methylene blue did not affect gel times under these conditions.

A similar experiment was performed using 4 arm NHS polyethylene glycol(molecular weight 10,000) mixed with an equimolar concentration of amultiarm amine-terminated polyethylene glycol (molecular weight 20,000).FD&C Blue #2 dye was present in the resultant hydrogel at aconcentration of 0.05, 0.1, 0.25, 0.5, 1.0, and 2.5 mg/ml. The hydrogelwas applied at a thickness of about 1.0 mm and observed by twoindependent observers and the ability to observe the gel was rated asadequate or inadequate. The results showed that good visualization ofthe hydrogel could be obtained at concentrations of at least 0.25 mg/mlFD&C Blue #2.

Example 23

This example was directed to the evaluation of the stability ofcolorants in solution. An 0.5 mg/ml amount of FD&C BLUE #2 dye (alsocalled indigo carmine) was dissolved in 0.1 M sodium borate decahydratepH 10 buffer, in deionized water, and in 0.01 M sodium phosphate bufferpH 4.0. Solutions were stored for up to 48 hours at 4° C., 25° C., and40° C. The dye appeared visually to be stable in solubility and color inthe distilled water and phosphate buffer solutions although the dyechanged the pH of the phosphate buffer from 4 to 6.9. The dye changedcolor in the borate solution. FD&C blue #2 and Methylene blue wereobserved to be not completely soluble so that their concentration issignificant. FD&C Blue #2 was soluble at less than 2.5 mg/ml but itsmaximum solubility was not ascertained.

Example 24

This experiment was directed to the effect of coloring agents ongelation times. A 4 arm NHS polyethylene glycol (molecular weight10,000) solution was mixed with an equimolar concentration of a multiarmamine. Both an amine terminated polyethylene glycol (molecular weight20,000) was evaluated as well as dilysine. Visualization agent was mixedwith the buffer used to reconstitute the amine and was present in theresultant hydrogel at a concentration of 12.5 mg/ml. Gel time tests wereperformed in triplicate. Gel time was measured immediately onreconstitution of the ester (time zero) and 1.5 hours later. The meangelation times in seconds±standard deviation were: FD&C Blue #1 gelationtime 1.57±0.12 time zero compared to 2.2±0.05 after 1.5 hours; FD&C Blue#2 gelation time 1.51±0.12 time zero compared to 2.08±0.09 after 1.5hours; Methylene Blue gelation time 1.67±0.28 at time zero compared to1.97±0.12 after 1.5 hours; No visualization agent gelation time1.39±0.02 compared to 1.78±0.13 after 1.5 hours. These visualizationagents did not cause an unacceptable change in gelation times.

While preferred illustrative embodiments of the invention are describedabove, it will be apparent to one skilled in the art that variouschanges and modifications may be made therein without departing from theinvention, and it is intended in the appended claims to cover all suchchanges and modifications which fall within the true spirit and scope ofthe invention.

1. A method for formulating a polymer composition that crosslinks toform a biodegradable hydrogel that is essentially completely degradablein vivo by hydrolytic degradation, the method comprising selecting aconcentration of visualization agent for the polymer composition so thatwhen the hydrogel is applied onto a substrate to reach an averagepredetermined thickness of the hydrogel, an observable change occursindicating the predetermined thickness of hydrogel has been deposited onthe substrate, wherein the hydrogel comprises chemical groups that areprone to aqueous hydrolysis and are degradable in vitro by exposure toaqueous solution.
 2. The method of claim 1 wherein the hydrogelcomprises a reaction product of a synthetic polymer that compriseselectrophilic functional groups and dilysine, trilysine, or tetralysine,wherein the reaction product is formed through the crosslinking betweenthe electrophilic functional groups of the synthetic polymer and theamino groups of the dilysine, trilysine, or tetralysine.
 3. The methodof claim 1 wherein the hydrogel comprises a reaction product of asynthetic polymer that comprises electrophilic functional groups and asynthetic polymer that comprises a plurality of primary amines orprimary thiols, wherein the reaction product is formed through thecrosslinking between the electrophilic functional groups of thesynthetic polymer and the plurality of primary amines or primary thiolsin the other synthetic polymer.
 4. The method of claim 1, wherein thevisualization agent is chosen from the group consisting of FD&C Blue #1,FD&C Blue #2, methylene blue, indocyanine green, visualization agentsthat provide a blue color, and visualization agents that provide a greencolor.
 5. The method of claim 1 wherein the visualization agent is notcovalently linked to the hydrogel.
 6. The method of claim 1 wherein thebiodegradable hydrogel is adherent to the substrate.
 7. The method ofclaim 1 wherein the hydrogel is free of amino acid sequences of morethan about four residues in number.
 8. The method of claim 1 wherein thepredetermined thickness is from about 0.5 mm to about 10.0 mm.
 9. Themethod of claim 1 wherein the polymer composition crosslinks to form ahydrogel within about 60 seconds after being applied to the substrate.10. The method of claim 1 wherein the hydrogel forms within 5 secondsafter contact with the substrate.
 11. The method of claim 1 wherein theobservable change is not being able to see the substrate tissue throughthe polymer composition, not being able to see patterns in the substratesurface through the polymer composition, the features of the substrateare obscured, or not being able to see the microvasculature on thesubstrate tissue.
 12. The method of claim 4 wherein the visualizationagent provides a blue color.
 13. The method of claim 11 wherein theobservable change is not being able to see through the polymercomposition.
 14. The method of claim 11 wherein the observable change isnot being able to see patterns in the substrate surface through thepolymer composition.
 15. The method of claim 11 wherein the observablechange is that the features of the substrate are obscured.
 16. Themethod of claim 11 wherein the observable change is not being able tosee the microvasculature on the substrate tissue.
 17. The method ofclaim 2 wherein the visualization agent provides a blue color.
 18. Themethod of claim 2 wherein the visualization agent is not covalentlylinked to the hydrogel.
 19. The method of claim 18 wherein thebiodegradable hydrogel is adherent to the substrate.
 20. The method ofclaim 19 wherein the polymer composition crosslinks to form a hydrogelwithin about 60 seconds after being applied to the substrate.
 21. Themethod of claim 2 wherein the hydrogel forms within 5 seconds aftercontact with the substrate.
 22. The method of claim 3 wherein thesynthetic polymer comprises the plurality of primary amines.
 23. Themethod of claim 22 wherein the visualization agent is not covalentlylinked to the hydrogel.
 24. The method of claim 23 wherein thebiodegradable hydrogel is adherent to the substrate.
 25. The method ofclaim 24 wherein the polymer composition crosslinks to form a hydrogelwithin about 60 seconds after being applied to the substrate.
 26. Themethod of claim 22 wherein the hydrogel forms within 5 seconds aftercontact with the substrate.
 27. The method of claim 3 wherein thevisualization agent is not covalently linked to the hydrogel.
 28. Themethod of claim 27 wherein the biodegradable hydrogel is adherent to thesubstrate.
 29. The method of claim 28 wherein the polymer compositioncrosslinks to form a hydrogel within about 60 seconds after beingapplied to the substrate.
 30. The method of claim 3 wherein the hydrogelforms within 5 seconds after contact with the substrate.